CN117089583A

CN117089583A - Method for recovering oil from fermentation product process and method for producing fermentation product

Info

Publication number: CN117089583A
Application number: CN202310897916.0A
Authority: CN
Inventors: J·马修斯; S·克拉克; J·江普; N·克里尔
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2013-06-24
Filing date: 2014-06-20
Publication date: 2023-11-21
Also published as: CN105339501A; WO2014209800A1; HUE057274T2; CA2915065A1; EP3013967A4; CA2915063C; CN116334149A; US11965143B2; EP3013968A4; US10844318B2; EP3572519A1; US20180362882A1; US20190338217A1; PL3013967T3; CA2915063A1; US10920172B2; US20230340361A1; ES2903010T3; US10093882B2; US10035973B2

Abstract

The present application relates to a process for recovering/extracting oil from a fermentation product production process based on starch-containing material, wherein an alpha-amylase, a high dose of protease, and optionally a glucoamylase is present and/or added during liquefaction. The application also relates to a process for producing a fermentation product, and to an enzyme composition suitable for use in the process of the application.

Description

Method for recovering oil from fermentation product process and method for producing fermentation product

Description of the divisional application

The application is a divisional application of Chinese patent application with the application date of 2014, month 06 and 20, application number of 201480035858X and the name of 'a method for recovering oil from a fermentation product process and a method for producing the fermentation product'.

Reference to sequence Listing

The present application encompasses a sequence listing in computer readable form. The computer readable form is incorporated herein by reference.

Technical Field

The present application relates to a method for recovering oil from a fermentation product production process, and a method for producing a fermentation product from starch-containing material. The application also relates to compositions suitable for use in the methods of the application.

Background

Fermentation products (e.g., ethanol) are typically produced by first milling starch-containing material during dry or wet milling, then degrading the material into fermentable sugars using enzymes, and finally converting the sugars directly or indirectly into the desired fermentation product using fermenting organisms. Liquid fermentation products are removed from the beer (often referred to as "beer") by, for example, distillation, which separates the desired fermentation product from other liquids and/or solids. The remainder is referred to as "stillage". The stillage is dewatered and separated into a solid phase and a liquid phase, for example, by centrifugation. This solid phase is called the "wet cake" (or "wet grain") and this liquid phase (supernatant) is called the "thin stillage". The wet cake and thin stillage contained about 35% and 7% solids, respectively. The dehydrated wet cake is dried to provide "distillers dried grains (Distillers Dried Grains)" (DDG) for use as a nutrient in animal feed. The thin stillage is typically evaporated to provide concentrate and slurry or alternatively may be recycled directly to the slurry tank as "backset". The concentrate may be forwarded to the methanator prior to discharge or may be recycled to the slurry tank. The slurry may be mixed into DDG or added to wet cake prior to drying to produce DDGS (distillers dried grains with solubles).

WO 2012/088303 (Novozymes) discloses a process for producing a fermentation product by liquefying starch-containing material at a pH ranging from 4.5 to 5.0, at a temperature ranging from 80 ℃ to 90 ℃, using a combination of an alpha-amylase having a T1/2 (min) of at least 10 at pH 4.5, 85 ℃, 0.12mm CaCl2 and a protease having a thermostability value (determined as relative activity at 80 ℃/70 ℃) of more than 20%.

WO 2013/08486 (novelin) discloses a process for producing a fermentation product by liquefying starch-containing material at a pH ranging from above between 5.0 and 7.0, at a temperature above the initial gelatinization temperature, using an alpha-amylase, a protease having a thermostability value of more than 20% (determined as relative activity at 80 ℃/70 ℃), and optionally an enzyme producing a carbohydrate source.

More and more ethanol plants extract oil from thin stillage and/or syrup as a byproduct for use in biodiesel production or other bio-renewable products. Much of the work in oil recovery/extraction in fermentation product production has been focused on improving the extractability of oil in thin stillage. Effective removal of oil is typically accomplished by hexane extraction. However, the use of hexane extraction is not widely used due to the high capital investment required. Thus, other methods have been explored to improve oil extraction from fermentation product production processes.

WO 2011/126897 (novelin) discloses a method of recovering oil by converting starch-containing material into dextrins with an alpha-amylase; saccharifying with an enzyme that produces a carbohydrate source to form a sugar; fermenting the sugars using a fermenting organism (wherein the fermentation medium comprises a hemicellulase); distilling the fermentation product to form distillers grains; separating the distillers grains into thin stillage and wet cake; and recovering oil from the thin stillage. The fermentation medium may further comprise a protease.

It is an object of the present invention to provide improved processes for increasing the amount of oil that can be recovered from a fermentation product production process, and to provide processes for producing fermentation products (e.g., ethanol) from starch-containing material that may provide higher fermentation product yields or other advantages over conventional processes.

Summary of The Invention

The present invention relates to a method for recovering/extracting oil from a fermentation product production process. The invention also relates to the production of a fermentation product (e.g., ethanol) from a starch-containing material in a process comprising liquefying the starch-containing material, saccharifying and fermenting the liquefied material. The invention also relates to compositions suitable for use in the methods of the invention.

In a first aspect, the present invention relates to a method for recovering/extracting oil from a fermentation product production process, the method comprising the steps of:

a) Liquefying the starch-containing material at a temperature above the initial gelatinization temperature using:

-an alpha-amylase;

-more than 0.5 microgram of pyrococcus furiosus protease per gram Dry Solids (DS);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied during and/or after step a); and/or

Downstream of the fermentation step c).

In one embodiment, between 0.5 and 100 micrograms of intense fireball protease per gram DS (dry solids) is present and/or added in liquefaction step a). In one embodiment, between 0.5 and 10 micrograms of intense fireball protease per gram DS (dry solids) is present and/or added in liquefaction step a). In one embodiment, between 1 and 50 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, between 1 and 10 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, between 1.5 and 5 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, about or more than 1.5 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, about or more than 2 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, about or more than 3 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a).

In a preferred embodiment, the thermolysin is the mature sequence shown in SEQ ID NO. 13 herein or one having at least 90% or 95% identity thereto.

Examples of alpha-amylases may be found in the "alpha-amylase present and/or added during liquefaction" section below.

Preferred alpha-amylases are those of the Bacillus species or variants thereof, particularly those derived from Bacillus stearothermophilus or Bacillus licheniformis.

In a preferred embodiment, the alpha-amylase is a double deleted Bacillus stearothermophilus alpha-amylase variant comprising a double deletion in position I181 x+G182 x (using SEQ ID NO:1 for numbering).

Preferred alpha-amylases include Bacillus stearothermophilus alpha-amylase variants, such as one of the following mutations shown in SEQ ID NO:1 herein:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

-I181 +g182 +n193F +v59A Q r+e129V +k177L +r179E +q254S +m284V; and

-I181 + g182 + N193F + E129V + K177L + R179E + K220P + N224L + s24q + Q254S (numbering using SEQ ID NO: 1).

In one embodiment, glucoamylase is present and/or added during liquefaction. Examples of suitable glucoamylases may be found in the "glucoamylase present and/or added during liquefaction" -section below.

In one embodiment, the glucoamylase has a thermostability of at least 80 ℃, preferably at least 82 ℃, such as at least 84 ℃, e.g., at least 86 ℃, e.g., at least 88 ℃ as determined by Differential Scanning Calorimetry (DSC) as described in example 3 below at pH 4.0.

In one embodiment, the glucoamylase has a thermal stability of at least 80 ℃, preferably at least 82 ℃, such as at least 84 ℃, e.g., at least 86 ℃, e.g., at least 88 ℃, e.g., at least 90 ℃ as determined by Differential Scanning Calorimetry (DSC) as described in example 3 below at a pH of 4.8.

Examples of particularly contemplated glucoamylases can be found in example 3 (table 6) below.

Preferred glucoamylases include Penicillium oxalicum glucoamylases, such as one having a K79V substitution as set forth in SEQ ID NO 14 herein and preferably further having one of the following:

-P11F+T65A+Q327F；

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

In a preferred embodiment, the liquefaction is carried out at a temperature between 80 ℃ and 90 ℃, for example about 85 ℃. In a preferred embodiment, the liquefaction is carried out at a pH in the range of pH above 5.0 to 6.0.

During saccharification and/or fermentation, glucoamylase is present and/or added. Examples of suitable glucoamylases may be found in the "glucoamylase" -sections which are present and/or added during saccharification and/or fermentation, infra.

In a second aspect, the present invention relates to methods for producing a fermentation product from starch-containing material, the methods comprising the steps of:

-an alpha-amylase;

-more than 2 micrograms of pneumococcal protease virulence per gram Dry Solids (DS);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

In a preferred embodiment, between 2 and 100 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In a preferred embodiment, between 2 and 10 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In a preferred embodiment, between 2.5 and 50. Mu.g of intense fireball protease per gram DS is present and/or added in liquefaction step a). In a preferred embodiment, between 2.5 and 10 micrograms/gram DS is present and/or added in liquefaction step a). In a preferred embodiment, between 2.5 and 5 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In a preferred embodiment, about or more than 3 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a).

In a preferred embodiment, the alpha-amylase is a double deleted Bacillus stearothermophilus alpha-amylase variant comprised in I181 x +G182 x (using SEQ ID NO:1 for numbering).

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

-I181 +g182 +n193F +v59A Q r+e129V +k177L +r179E +q254S +m284V; and

-P11F+T65A+Q327F；

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

In a third aspect, the present invention relates to an enzyme composition comprising:

(i) Bacillus species alpha-amylase, or a variant thereof;

(ii) A strong pyrococcus protease;

wherein the ratio between alpha-amylase and protease is in the range from 1:1 and 1:25 (micrograms of alpha-amylase: micrograms of protease).

In a preferred embodiment, the alpha-amylase is a double deleted Bacillus stearothermophilus alpha-amylase variant comprised in I181 x +G182 x (numbered using SEQ ID NO: 1).

Preferred alpha-amylases include Bacillus stearothermophilus alpha-amylase variants, such as those shown in SEQ ID NO. 1 herein, having the following mutations:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

-i181+g182+n193 f+v59aq9r+e129 v+k177l+r179e+q254s+m284V; and

Preferred glucoamylases include Penicillium oxalicum glucoamylases, such as one with a K79V substitution shown in SEQ ID NO 14 herein and further with one of the following:

-P11F+T65A+Q327F；

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

In a preferred embodiment, the ratio between alpha-amylase and protease is in the range of between 1:1.2 and 1:10, for example about 1:1.4 (micrograms of alpha-amylase: micrograms of protease).

In another embodiment, the enzyme composition of the invention comprises glucoamylase and the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase: micrograms of glucoamylase).

Brief Description of Drawings

FIG. 1 shows the comparison (no statistical difference) of oil extraction between protease Pfu (1.5. Mu.g/gDS) and protease X.

FIG. 2 shows the comparison (statistical difference) of oil extraction between protease Pfu (3. Mu.g/gDS) and protease X.

FIG. 3 shows the comparison (statistical difference) of oil extraction between protease Pfu (5. Mu.g/gDS) and protease X.

FIG. 4 shows the ethanol concentration (% w/v) of urea-free fermentation.

FIG. 5 shows a one-way analysis of the comparison of ethanol concentration (% w/v) without urea fermentation for the 0, 1.5, 3 and 5. Mu.g/gDS proteases Pfu.

FIG. 6 shows the ethanol concentration (% w/v) of fermentations operated with 200ppm urea for protease X added to SSF and protease Pfu (1.5, 3 and 5. Mu.g/gDS).

FIG. 7 shows a one-way analysis of ethanol concentration (% w/v) comparisons for 0, 1.5, 3 and 5. Mu.g/gDS protease Pfu for fermentation based on 200ppm urea.

FIG. 8 shows the glycerol concentration (% w/v) for 54 hours. The highest dose of protease Pfu (5. Mu.g/gDS) was about 10% lower than the control for protease X.

FIG. 9 shows the ethanol concentration (% w/v) after 54 hours when protease Pfu from 0 (control) to 50. Mu.g/gDS was added during liquefaction.

FIG. 10 shows the glycerol concentration (% w/v) after 54 hours when protease Pfu from 0 (control) to 50. Mu.g/gDS was added during liquefaction.

FIG. 11 shows the glucose concentration (% w/v) after 54 hours when protease Pfu from 0 (control) to 50. Mu.g/gDS was added during liquefaction.

Detailed Description

The present invention relates to a method for recovering oil from a fermentation product production process, and a method for producing a fermentation product from starch-containing material. The invention also relates to compositions suitable for use in the methods of the invention.

The present invention has found that when alpha-amylase, high doses of intense fireball protease and optionally glucoamylase are combined, an increased amount of oil can be recovered downstream of liquefaction or fermentation, as compared to when alpha-amylase is added during liquefaction and protease is added during fermentation (SSF).

The present invention also found that increased ethanol yields are obtained when alpha-amylase, greater than 2 micrograms of intense firecoccus protease per gram of Dry Solids (DS) and glucoamylase are combined, as compared to when alpha-amylase, less than 2 micrograms of intense firecoccus protease per gram of Dry Solids (DS) and glucoamylase are used in the liquefaction process.

The present invention also found that the glycerol concentration with protease Pfu (5. Mu.g/gDS) was lower than with the addition of protease in SSF.

It has also been found that the ethanol process of the present invention can be operated effectively with or without the addition of a nitrogen source (e.g., urea) to the SSF.

The method for recovering/extracting oil of the present invention

In a first aspect, the present invention relates to a method for recovering oil from a fermentation product production process, the method comprising the steps of:

-an alpha-amylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

In one embodiment, the oil is recovered/extracted during and/or after liquefying the starch-containing material. In one embodiment, the oil is recovered from the stillage. In one embodiment, the oil is recovered from thin stillage. In one embodiment, the oil is recovered from the slurry.

In one embodiment, between 0.5 and 100 micrograms of intense fireball protease per gram DS (dry solids) is present and/or added in liquefaction step a). In one embodiment, between 0.5 and 10 micrograms of intense fireball protease per gram DS (dry solids) is present and/or added in liquefaction step a). In one embodiment, between 1 and 50 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, between 1 and 10 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, between 1.5 and 5 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, about or more than 1 microgram of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, about or more than 1.5 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a). In one embodiment, about or more than 2 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a).

In one embodiment, between 2-100 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, between 2.5-50 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, between 2.5-10 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, between 2.5-5 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, between 2.75-50 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, between 2.75-10 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, between 2.75-5 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In a preferred embodiment, about or more than 3 micrograms of intense fireball protease per gram DS is present and/or added in liquefaction step a).

In a preferred embodiment, the thermolysin is the mature sequence shown in SEQ ID NO. 13 herein. In one embodiment, the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

In one embodiment, in steps a) -c), for example in saccharification step b), fermentation step c), or Simultaneous Saccharification and Fermentation (SSF), no nitrogen compounds (e.g. urea) are present and/or added.

In one embodiment, in steps a) -c), for example in saccharification step b) or fermentation step c) or Simultaneous Saccharification and Fermentation (SSF), 10-1,000ppm, for example 50-800ppm, for example 100-600ppm, for example 200-500ppm, of nitrogen compounds, preferably urea, are present and/or added.

Method for producing a fermentation product according to the invention

-an alpha-amylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

In one embodiment, the fermentation product is recovered after fermentation. In a preferred embodiment, the fermentation product is recovered after fermentation (e.g., by distillation). In one embodiment, the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol and/or industrial ethanol.

In one embodiment, 2-100 micrograms of virulent pneumococcal protease per gram DS is added and/or present during liquefaction. In one embodiment, 2.5-50 micrograms of pneumococcal protease per gram DS is added and/or present during liquefaction. In one embodiment, 2.5-10 micrograms of pneumococcal protease per gram DS is added and/or present during liquefaction. In one embodiment, 2.5-5 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In one embodiment, 2.75-50 microgram of pneumococcal protease per gram DS is added and/or present during liquefaction. In one embodiment, 2.75-10 micrograms of pneumococcal protease per gram DS is added and/or present during liquefaction. In one embodiment, 2.75-5 micrograms of intense fireball protease per gram DS is added and/or present during liquefaction. In a preferred embodiment, about 3 micrograms of intense firefly protease per gram DS is present and/or added in liquefaction step a).

In a preferred embodiment, the thermolysin is one of SEQ ID NO. 13 as set forth herein. In one embodiment, the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

In a preferred embodiment, in steps a) -c), no nitrogen compounds are present and/or added, for example during saccharification step b) or fermentation step c) or Simultaneous Saccharification and Fermentation (SSF).

In one embodiment, in steps a) -c), for example in saccharification step b) or fermentation step c) or Simultaneous Saccharification and Fermentation (SSF), 10-1,000ppm, for example 50-800ppm, for example 100-600ppm, for example 200-500ppm of nitrogen compound (preferably urea) is present and/or added.

Alpha-amylase present and/or added during liquefaction

The alpha-amylase added during liquefaction step a) in the process of the invention (i.e. the oil recovery process and the fermentation product production process) may be any alpha-amylase.

Preferably a bacterial alpha-amylase, which is typically stable at the temperatures used in the liquefaction process.

In one embodiment, the alpha-amylase is a strain from the genus bacillus.

In a preferred embodiment, the alpha-amylase is derived from a strain of Bacillus stearothermophilus (e.g., the sequence shown in SEQ ID NO: 1). In one embodiment, the alpha-amylase is a Bacillus stearothermophilus alpha-amylase as set forth in SEQ ID NO. 1 herein, e.g., one having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO. 1 herein.

In one embodiment, the Bacillus stearothermophilus alpha-amylase or variant thereof is truncated (preferably at the C-terminus), preferably truncated to have about 491 amino acids, e.g., from 480 to 495 amino acids.

In one embodiment, the Bacillus stearothermophilus alpha-amylase has a double deletion at position I181+G182 and optionally has an N193F substitution (numbered using SEQ ID NO: 1).

In another embodiment, the Bacillus stearothermophilus alpha-amylase has a double deletion at position R179+G180 and optionally has an N193F substitution (numbered using SEQ ID NO: 1).

In one embodiment, the bacillus stearothermophilus alpha-amylase has a substitution at position S242, preferably the S242Q substitution.

In one embodiment, the Bacillus stearothermophilus alpha-amylase has a substitution at position E188, preferably an E188P substitution.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 10.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 15.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 20.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 25.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 30.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 40.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 50.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 60. In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ T1 +.sub.10-70 below2(min)。

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 15 and 70.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 20 and 70.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 25 and 70.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 30 and 70.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 40 and 70.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 50 and 70.

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has T1/2 (min) between 60 and 70.

In one embodiment, the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants having the following mutations in addition to I181 x+g182 x and optionally N193F:

in a preferred embodiment, the alpha-amylase is selected from the group of Bacillus stearothermophilus alpha-amylase variants:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

-i181+g182+n193 f+v59aq9r+e129 v+k177l+r179e+q254s+m284V; and

According to the invention, the alpha-amylase variant has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ ID NO. 1 herein.

In another embodiment, the alpha-amylase is a Bacillus licheniformis alpha-amylase, or a variant thereof. In one embodiment, the Bacillus licheniformis alpha-amylase is one of the SEQ ID NOs shown herein as 21. According to the invention, the alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g. even at least 96%, at least 97%, at least 98%, at least 99% identity to the mature part of the polypeptide of SEQ ID NO. 21 herein.

According to the invention, the alpha-amylase may be present and/or added at a concentration of 0.1-100 micrograms/gram DS, e.g., 0.5-50 micrograms/gram DS, e.g., 1-25 micrograms/gram DS, e.g., 1-10 micrograms/gram DS, e.g., 2-5 micrograms/gram DS.

In one embodiment, from 1 to 10 micrograms of the thermolysin and 1 to 10 micrograms of the bacillus stearothermophilus alpha-amylase are present and/or added during liquefaction.

Glucoamylase present and/or added during liquefaction

In one embodiment, in liquefaction step a) in the process of the invention (i.e. the oil recovery process and the fermentation product production process), glucoamylase is present and/or added.

In a preferred embodiment, the glucoamylase present and/or added during liquefaction is determined to have a thermostability of at least 20%, e.g. at least 30%, preferably at least 35% at 85 ℃, pH 5.3 as disclosed in example 2 herein or in example 8 in WO 2011/127802.

In one embodiment, the glucoamylase has a relative activity of at least 90%, preferably at least 95%, preferably at least 97% determined as disclosed in this example 2 or in example 8 of WO 2011/127802 at an optimal pH of pH 5.0.

In one embodiment, the glucoamylase has a pH stability of at least 80%, at least 85%, at least 90% as determined in example 2 herein or in example 8 of WO 2011/127802 at pH 5.0.

In a preferred embodiment, the glucoamylase present and/or added in liquefaction step a) is derived from a penicillium strain, in particular a penicillium oxalate strain as disclosed in SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 9 or 14 herein.

In one embodiment, the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 9 or 14 herein.

In a preferred embodiment, the glucoamylase is a variant of the penicillium oxalicum glucoamylase shown in WO 2011/127802 as SEQ ID No. 2 or in SEQ ID No. 14 herein, having a K79V substitution (numbered using the mature sequence shown in SEQ ID No. 14), such as the variant disclosed in WO 2013/053801 (hereby incorporated by reference).

In one embodiment, the penicillium oxalicum glucoamylase has a K79V substitution (numbered using SEQ ID NO: 14), and preferably further has one of the following substitutions:

T65A; or (b)

Q327F; or (b)

E501V; or (b)

Y504T; or (b)

Y504; or (b)

t65a+q327F; or (b)

t65a+e501V; or (b)

t65a+y504T; or (b)

T65a+y504; or (b)

Q327F+E501V; or (b)

Q327F+Y504T; or (b)

Q327 f+y504; or (b)

E501V+Y504T; or (b)

E501 v+y504; or (b)

T65a+q327f+e501V; or (b)

T65a+q327f+y504T; or (b)

T65a+e501v+y504T; or (b)

Q327F+E501V+Y504T; or (b)

T65a+q327 f+y504; or (b)

T65a+e501 v+y504; or (b)

Q327f+e501 v+y504; or (b)

T65a+q327f+e501v+y504T; or (b)

T65A+Q327F+E501V+Y504*；

E501V+Y504T; or (b)

t65a+k161S; or (b)

t65a+q405T; or (b)

t65a+q327W; or (b)

t65a+q327F; or (b)

t65a+q327Y; or (b)

p11f+t65a+q327F; or (b)

R1k+d3w+k5q+g7v+n8s+t10k+p1s+t65a+q 327F; or (b)

p2n+p4s+p11f+t65a+q327F; or (b)

p11f+d26c+k33c+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

R1e+d3n+p4g+g6r+g7a+n8a+t10d+p11d+t65a+q327F; or (b)

p11f+t65a+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P11f+t65a+q327w+e501v+y504T; or (b)

T65a+q327f+e501v+y504T; or (b)

t65a+s105p+q327W; or (b)

t65a+s105p+q327F; or (b)

T65a+q327w+s364P; or (b)

T65a+q327f+s364P; or (b)

t65a+s103n+q327F; or (b)

P2n+p4s+p11f+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447S; or (b)

P2n+p4s+p11f+t65a+i172v+q327F; or (b)

P2n+p4s+p11f+t65a+q327 f+n502; or (b)

P2n+p4s+p11f+t65a+q327f+n502t+p563s+k571E; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+n564d+k571S; or (b)

P2n+p4s+p11f+t65a+q327f+s377t; or (b)

P2n+p4s+p11f+t65a+v325t+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+i172v+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s377t+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+i375a+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k21a+k217d+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

P2n+p4s+t10d+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+f12y+t65a+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t568N; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+k524t+g526A; or (b)

P2n+p4s+p11f+k34y+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+f80+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k12s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+n502 t+y504; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+k9a+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9g+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9i+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+l72v+q327f+e501v+y504T; or (b)

S255n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+e7n+v79 k+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+g220n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+y245n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q257n+q7f+e501 v+y504T; or (b)

P2n+p4s+p11f+t65a+d279n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s359n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+d370n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460t+p468t+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+t463n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s465n+e501v+y504T; or (b)

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T。

In a preferred embodiment, the glucoamylase present and/or added during liquefaction is a penicillium oxalicum glucoamylase having a K79V substitution and preferably further having one of the following substitutions:

-P11F+T65A+Q327F；

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

In one embodiment, the glucoamylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 14 herein.

The glucoamylase may be added in an amount of from 0.1 to 100 micrograms of EP/g, e.g., 0.5 to 50 micrograms of EP/g, e.g., 1 to 25 micrograms of EP/g, e.g., 2 to 12 micrograms of EP/g DS.

Glucoamylase present and/or added in saccharification and/or fermentation

In the process of the invention (i.e., the oil recovery process and the fermentation product production process), glucoamylase is present and/or added during saccharification and/or fermentation, preferably Simultaneous Saccharification and Fermentation (SSF).

In one embodiment, the glucoamylase present and/or added during saccharification and/or fermentation is of fungal origin, preferably from a strain of aspergillus, preferably a strain of aspergillus niger, aspergillus awamori, or aspergillus oryzae; or a strain of Trichoderma, preferably a strain of Trichoderma reesei; or a strain of the genus Penicillium, preferably a strain of the genus Emerson Penicillium.

In one embodiment, the glucoamylase is derived from a strain of a genus Penicillium, such as Emerson's Penicillium (e.g., one of SEQ ID NOs: 19 shown herein).

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 19 herein;

(ii) A glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID No. 19 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Mitigation, in particular the Hamiltonia haemolytica strain described in WO 2011/066576 (SEQ ID NO 2, 4 or 6), for example one of SEQ ID NO 4 or SEQ ID NO 18 herein shown in WO 2011/066576.

In one embodiment, the glucoamylase is derived from a Phlebsiella strain, e.g., phlebia portentosa or Phlebia portentosa strain, in particular a Phlebia portentosa strain (SEQ ID NO:2, 4, 6, 8, 10, 12, 14 or 16) as described in WO 2011/068803. In a preferred embodiment, the glucoamylase is SEQ ID NO. 2 shown in WO 2011/068803 or Philippine in SEQ ID NO. 15 herein.

In a preferred embodiment, the glucoamylase is derived from a Myxophyllum treponema (e.g., one of SEQ ID NOs: 15 shown herein). In one embodiment, the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 15 herein;

(ii) A glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID No. 15 herein.

In another preferred embodiment, the glucoamylase is derived from Philippine (e.g., one of SEQ ID NOs: 17 as set forth herein). In one embodiment, the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 17 herein;

(ii) A glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID No. 17 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Nigrothomes (Nigrothomes), in particular the strain of the genus Nigrothomes (SEQ ID NO: 2) disclosed in WO 2012/064351 (all references hereby incorporated by reference).

In one embodiment, the glucoamylase may be added to saccharification and/or fermentation in the following amounts: 0.0001-20AGU/g DS, preferably 0.001-10AGU/g DS, especially between 0.01-5AGU/g DS, e.g. 0.1-2AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN (storage area network) ^TM SUPER、SAN ^TM EXTRAL、SPIRIZYME ^TM PLUS、SPIRIZYME ^TM FUEL、SPIRIZYME ^TM B4U、SPIRIZYME ^TM ULTRA、SPIRIZYME ^TM EXCEL and AMG ^TM E (from Novozymes A/S); OPTIDEX ^TM 300. GC480, GC417 (from dupont (dupont)); AMIGASE (AMIGASE) ^TM And AMIGASE ^TM PLUS (from DSM company); G-ZYME ^TM G900、G-ZYME ^TM And G990 ZR (from DuPont).

According to a preferred embodiment of the invention, the glucoamylase is present in combination with and/or added to the alpha-amylase during saccharification and/or fermentation. Examples of suitable alpha-amylases are described below.

Alpha-amylase present and/or added in saccharification and/or fermentation

In one embodiment, an alpha-amylase is present and/or added during saccharification and/or fermentation in the methods of the invention. In a preferred embodiment, the alpha-amylase is of fungal or bacterial origin. In a preferred embodiment, the alpha-amylase is a fungal acid stable alpha-amylase. Fungal acid stable alpha-amylases are alpha-amylases having activity in the pH range of 3.0 to 7.0, and preferably in the range of 3.5 to 6.5, including activity at pH of about 4.0, 4.5, 5.0, 5.5, and 6.0.

In a preferred embodiment, the alpha-amylase present and/or added in saccharification and/or fermentation is derived from a strain of rhizomucor, preferably a strain of rhizomucor minium, such as one of SEQ ID NO:3 shown in WO 2013/006756, such as a rhizomucor minium alpha-amylase hybrid with an Aspergillus niger linker and a starch binding domain, such as one of SEQ ID NO:16 shown herein, or a variant thereof.

In one embodiment, the alpha-amylase present and/or added in saccharification and/or fermentation is selected from the group consisting of:

(i) An alpha-amylase comprising the mature polypeptide of SEQ ID NO. 16 herein;

(ii) An alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID No. 16 herein.

In a preferred embodiment, the alpha-amylase is an alpha-amylase variant shown in SEQ ID NO 13 having at least one of the following substitutions or combinations: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; s123h+y141W; g20s+y141W; a76g+y141W; g128d+y141W; g128d+d143N; p219C+Y141W; n142d+d143N; y141w+k192R; y141w+d143N; y141w+n383R; y141w+p219c+a265C; y141 w+n517d+d143N; y141w+k192R V410A; g128d+y141w+d143N; y141w+d143n+p219C; y141w+d143n+k192R; g128d+d143n+k192R; y141w+d143 n+k168r+p219C; g217d+y141 w+d143n+k192R; or G128D+Y141W+D143N+K192R+P219C (numbered with SEQ ID NO: 16).

In one embodiment, the alpha-amylase is derived from Rhizomucor minutissima having an Aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD), preferably as disclosed herein as SEQ ID NO 13, preferably with one or more of the following substitutions: G128D, D N, preferably G128D+D143N (numbered using SEQ ID NO: 13).

In one embodiment, the alpha-amylase variant present and/or added in saccharification and/or fermentation has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID NO. 16 herein.

In one embodiment, the alpha-amylase is derived from a strain of Aspergillus, such as Aspergillus niger (e.g., one shown as SEQ ID NO:9 in U.S. Pat. No. 8,048,657); or Aspergillus kawachii (e.g., one shown as SEQ ID NO:5 in U.S. Pat. No. 8,048,657).

In one embodiment, the alpha-amylase is derived from a Trichoderma reesei strain, e.g., one of SEQ ID NOs 13 as shown in U.S. Pat. No. 8,048,657.

In a preferred embodiment, the ratio between glucoamylase and alpha-amylase present and/or added during saccharification and/or fermentation may preferably be in the range of 500:1 to 1:1, e.g. from 250:1 to 1:1, e.g. from 100:1 to 1:1, e.g. from 100:2 to 100:50, e.g. from 100:3 to 100:70.

Pullulanase present and/or added in liquefaction and/or saccharification and/or fermentation

Pullulanase may be present and/or added during liquefaction step a) and/or saccharification step b) or fermentation step c) or simultaneous saccharification and fermentation.

Pullulanases (e.c. 3.2.1.41, pullulan 6-glucan-hydrolase) are debranching enzymes, which are characterized by their ability to hydrolyze alpha-1, 6-glycosidic bonds in, for example, amylopectin and amylopectin.

Pullulanases contemplated according to the present invention include pullulanases from Bacillus amyloliquefaciens (Bacillus amyloderamificans) disclosed in U.S. Pat. No. 4,560,651 (hereby incorporated by reference), pullulanases disclosed as SEQ ID NO. 2 in WO 01/151620 (hereby incorporated by reference), pullulanases from Bacillus debranching (Bacillus deramificans) disclosed as SEQ ID NO. 4 in WO 01/151620 (hereby incorporated by reference), and pullulanases from Bacillus acidophilus disclosed as SEQ ID NO. 6 in WO 01/151620 (hereby incorporated by reference), and also pullulanases described in FEMS microbiology communication (FEMS Mic. Let.) (1994) 115,97-106.

Additional pullulanases contemplated according to the present invention include pullulanases from Thermococcus Wo Sishi, in particular from Thermococcus Wo Sishi DSM No.3773 disclosed in WO 92/02614.

In one embodiment, the pullulanase is a GH57 family pullulanase, wherein the pullulanase preferably comprises an X47 domain as disclosed in WO 2011/087836. More specifically, the pullulanase may be derived from strains from the genus thermophilic cocci, including thermophilic and hydrothermal pyrococci (Thermococcus hydrothermalis), or hybrids thereof. In one embodiment, the pullulanase is a Thermococcus bifidus pullulanase truncated at position X4 or a Thermococcus bifidus/Thermococcus hybrid having a truncation position X4, disclosed in WO 2011/087836 or shown in SEQ ID NO. 12 herein.

In another embodiment, the pullulanase is one comprising the X46 domain disclosed in WO 2011/076123 (novelin).

According to the invention, the pullulanase may be added in an effective amount, comprising a preferred amount of about 0.0001-10mg enzyme protein per gram DS, preferably 0.0001-0.10mg enzyme protein per gram DS, more preferably 0.0001-0.010mg enzyme protein per gram DS. Pullulanase activity can be determined as NPUN. The assays for determining NPUN are described in the materials and methods section below.

Suitable commercially available pullulanase products include PROMOZYME D, PROMOZYME ^TM D2 (Norwechat, denmark), OPTIMAX L-300 (Jieraceae, USA), AMANO 8 Annean, japan).

Additional aspects of the methods of the invention

Prior to liquefaction step a), the process of the present invention (including the process of extracting/recovering oil and the process for producing fermentation products) may comprise the steps of:

i) Reducing the particle size of the starch-containing material, preferably by dry milling;

ii) forming a slurry comprising the starch-containing material and water.

In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material is suitable for passing through a screen having a #6 screen.

In one embodiment, the pH during liquefaction is between above 4.5-6.5, such as 4.5-5.0, such as about 4.8, or between 5.0-6.2, such as 5.0-6.0, such as between 5.0-5.5, such as about 5.2, such as about 5.4, such as about 5.6, such as about 5.8.

In one embodiment, the temperature during liquefaction is higher than the initial gelatinization temperature, preferably in the range from 70 ℃ to 100 ℃, e.g. between 75 ℃ and 95 ℃, e.g. between 75 ℃ and 90 ℃, preferably between 80 ℃ and 90 ℃, especially about 85 ℃.

In one embodiment, a jet cooking step is performed prior to liquefaction in step a). In one embodiment, the jet cooking is carried out at a temperature of between 110 ℃ and 145 ℃, preferably 120 ℃ and 140 ℃, for example 125 ℃ and 135 ℃, preferably about 130 ℃ for about 1 to 15 minutes, preferably about 3 to 10 minutes, especially about 5 minutes or so.

In a preferred embodiment, saccharification and fermentation are performed sequentially or simultaneously.

In one embodiment, saccharification is conducted at a temperature from 20 ℃ to 75 ℃, preferably from 40 ℃ to 70 ℃, for example about 60 ℃, and at a pH between 4 and 5.

In one embodiment, fermentation or Simultaneous Saccharification and Fermentation (SSF) is conducted at a temperature of from 25 ℃ to 40 ℃, e.g., from 28 ℃ to 35 ℃, e.g., from 30 ℃ to 34 ℃, preferably about 32 ℃. In one embodiment, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.

In a preferred embodiment, the fermentation product is recovered after fermentation, for example by distillation.

In one embodiment, the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol and/or industrial ethanol.

In one embodiment, the starch-containing starting material is whole grain. In one embodiment, the starch-containing material is selected from the group consisting of: corn, wheat, barley, rye, milo, sago, tapioca (casstra), manioc, tapioca, sorghum, rice, and potato.

In one embodiment, the fermenting microorganism is a yeast, preferably a Saccharomyces strain, especially a Saccharomyces cerevisiae strain.

In one embodiment, the alpha-amylase is a bacterial or fungal alpha-amylase.

In one embodiment, saccharification step b) and fermentation step c) are performed simultaneously or sequentially.

In one embodiment, the temperature in step (a) is higher than the initial gelatinization temperature, e.g. at a temperature between 80 ℃ and 90 ℃, e.g. about 85 ℃.

In one embodiment, the process of the present invention further comprises a pre-saccharification step, carried out at a temperature between 30-65 ℃ for 40-90 minutes, prior to saccharification step b). In one embodiment, saccharification is conducted at a temperature from 20 ℃ to 75 ℃, preferably from 40 ℃ to 70 ℃, for example about 60 ℃, and at a pH between 4 and 5. In one embodiment, fermentation step c) or Simultaneous Saccharification and Fermentation (SSF) (i.e. steps b) and c)) is performed at a temperature of from 25 ℃ to 40 ℃, e.g. from 28 ℃ to 35 ℃, e.g. from 30 ℃ to 34 ℃, preferably around 32 ℃. In one embodiment, the fermentation step c) or Simultaneous Saccharification and Fermentation (SSF) (i.e. steps b) and c)) is carried out for 6 to 120 hours, in particular 24 to 96 hours.

In one embodiment, the separation in step e) is by centrifugation, preferably a decanting centrifuge, filtration, preferably using a filter press, screw press, plate and frame press, gravity concentrator or dehydrator.

In one embodiment, the fermentation product is recovered by distillation.

Examples of embodiments of the specific methods of the invention

Oil recovery:

in a preferred embodiment, the present invention relates to a method of recovering oil, the method comprising the steps of:

bacillus stearothermophilus alpha-amylase comprising a double deletion at position I181+G182 (numbered using SEQ ID NO: 1);

-the penicillium oxalate comprising a K79V substitution shown in SEQ ID No. 14;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

-a bacillus stearothermophilus alpha-amylase comprising: the following substitutions N193 F+V59A+Q9R+E129 V+K177L+R179E+Q254S+M284V (numbering using SEQ ID NO: 1) were double deleted and truncated to 491 amino acids at position I181+G182.

-a penicillium oxalicum glucoamylase having the following mutations: k79 V+P2N+P4S+P1F+T65A+Q327F (numbered using SEQ ID NO: 14);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

In a preferred embodiment, the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms alpha-amylase/g DS: micrograms glucoamylase/g DS).

In a preferred embodiment, the ratio between alpha-amylase and protease during liquefaction is between 1:1 and 1:25, e.g. in the range between 1:1.2 and 1:10, e.g. about 1:1.4 (micrograms of alpha-amylase/gram of DS: micrograms of protease/gram of DS).

Producing a fermentation product:

in a preferred embodiment, the present invention relates to methods for producing a fermentation product from starch-containing material, comprising the steps of:

-an alpha-amylase derived from bacillus stearothermophilus;

-more than 2 micrograms of pneumococcal protease virulence per gram Dry Solids (DS); and

-optionally a penicillium oxalicum glucoamylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

-an alpha-amylase, preferably derived from bacillus stearothermophilus, at pH 4.5, 85 ℃, 0.1 2mM CaCl ₂ T1/2 (min) with at least 10;

-optionally a glucoamylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

an alpha-amylase, preferably derived from Bacillus stearothermophilus, at pH 4.5, 85℃and 0.12mM CaCl ₂ T1/2 (min) with at least 10;

-a penicillium oxalicum glucoamylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

-an alpha-amylase derived from bacillus stearothermophilus having a double deletion at position I181+ G182 and optionally a substitution N193F; additionally one of the following substitution sets:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

E129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

-a penicillium oxalicum glucoamylase of SEQ ID No. 14 having a substitution selected from the group consisting of:

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbered with SEQ ID NO: 14);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

a) Liquefying starch-containing material at a temperature between 80 ℃ and 90 ℃ at a pH ranging from above between 4.5 and 6.5 using:

-an alpha-amylase derived from bacillus stearothermophilus having double deletions of I181+ G182, optionally substituted with N193F; and additionally one of the following substitution sets:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

Greater than 2 micrograms, for example between 2 and 5 micrograms, preferably about or greater than 3 micrograms of intense fireball protease per gram of DS Dry Solids (DS);

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbered with SEQ ID NO: 14);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

-an alpha-amylase derived from bacillus stearothermophilus having double deletions of I181+ G182 to replace N193F; and additionally one of the following substitution sets:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

Greater than 2 micrograms, for example between 2 and 5 micrograms, preferably about or greater than 3 micrograms of intense fireball protease per gram of DS Dry Solids (DS)

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbered with SEQ ID NO: 14);

b) Using a Rhizomucor minibus glucoamylase having an Aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD), preferably as disclosed herein as SEQ ID NO 13, preferably with one or more of the following substitutions: G128D, D N, preferably G128D+D143N (numbered with SEQ ID NO: 13) for saccharification;

c) Fermentation is performed using a fermenting organism.

In one embodiment, the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms alpha-amylase/g DS: micrograms glucoamylase/g DS).

In one embodiment, the ratio between alpha-amylase and protease in liquefaction is between 1:1 and 1:25, e.g., in the range between 1:1.2 and 1:10, e.g., about 1:1.4 (micrograms of alpha-amylase/g DS: micrograms of protease/gram DS).

Fermentation medium

The environment in which the fermentation is carried out is commonly referred to as the "fermentation medium (fermentation media or fermentation medium)". The fermentation medium comprises a fermentation substrate, i.e. a carbohydrate source that is metabolized by the fermenting organism. According to the invention, the fermentation medium may comprise nutrients for one or more fermenting organisms and one or more growth stimulators. Nutrients and growth stimulators are widely used in the fermentation field and include nitrogen sources such as ammonia, urea, vitamins and minerals or combinations thereof.

Fermenting organism

The term "fermenting organism" refers to any organism suitable for use in a fermentation process and capable of producing a desired fermentation product, including bacterial and fungal organisms, particularly yeast. Particularly suitable fermenting organisms are capable of fermenting, i.e. converting, sugar (e.g. glucose or maltose) directly or indirectly into, i.e. into, a desired fermentation product (e.g. ethanol). Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeasts include Saccharomyces strains, in particular Saccharomyces cerevisiae.

The concentration of suitable living fermenting organisms during fermentation, such as SSF, is well known in the art or can be readily determined by one of ordinary skill in the art. In one embodiment, the fermenting organism (e.g., ethanol fermenting yeast (e.g., saccharomyces cerevisiae)) is added to the fermentation medium such that the viable fermenting organism (e.g., yeast) count per mL of fermentation medium ranges from 10 ⁵ To 10 ¹² Preferably from 10 ⁷ To 10 ¹⁰ In particular about 5x10 ⁷ And each.

Examples of commercially available yeasts include, for example, RED STAR ^TM And ETHANOL RED ^TM Yeast (obtainable from Fusarium/Le Sifu (Fermentis/Lesafre), FALI (obtainable from Fleischmann's Yeast), SUPERSTART and THERMOSAC ^TM Fresh yeasts (available from ethanol technologies (Ethanol Technology), wisconsin (WI), U.S.), BIOFERM AFT and XR (available from NABC-North American Bioproduct group (NABC-North American Bioproducts Corporation), georgia (GA), U.S.), GERT STRAND (available from Gert Strand AB, sweden) and FERMINOL (available from Dissman food ingredients (DSM Specialties)).

Starch-containing material

Any suitable starch-containing material may be used in accordance with the present invention. The starting materials are generally selected based on the desired fermentation product. Examples of starch-containing materials suitable for use in the methods of the invention include whole grains, corn, wheat, barley, rye, milo (sago), sago, tapioca, sorghum, rice, pea, legume, or sweet potato, or mixtures thereof, or starches derived therefrom, or cereals. Waxy and non-waxy types of corn and barley are also contemplated. In a preferred embodiment, the starch-containing material used for ethanol production according to the present invention is corn or wheat.

Fermentation product

The term "fermentation product" means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (e.g., ethanol, methanol, butanol; polyols such as glycerol, sorbitol, and inositol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gas (e.g. H ₂ And CO ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the Antibiotics (e.g., penicillin and tetracycline); an enzyme; vitamins (e.g. riboflavin, B) ₁₂ Beta-carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, such as fuel ethanol; drinking ethanol, i.e. drinking ethanol; or industrial ethanol or products for the consumer alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. Preferred beer types include ale (ale), stout, porter, lager, bitter, malt (malt liqur), low malt (happouhu), high alcohol beer, low calorie beer, or light beer. Preferably, the process of the present invention is for the production of an alcohol (e.g. ethanol). The fermentation product obtained according to the invention, e.g. ethanol, may be used as fuel, typically blended with gasoline. However, in the case of ethanol it can also be used as drinking ethanol.

Recovery of fermentation products

After fermentation, or SSF, the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (ethanol). Alternatively, the desired fermentation product may be extracted from the fermentation medium by microfiltration or membrane filtration techniques. The fermentation product may also be recovered by steam stripping or other methods well known in the art.

Recovery of oil

According to the invention, oil is recovered from the stillage, from the thin stillage, or from the slurry during and/or after liquefaction. The oil may be recovered by extraction. In one embodiment, the oil is recovered by hexane extraction. Other oil recovery techniques well known in the art may also be used.

The enzyme composition of the present invention

The enzyme composition of the invention comprises an alpha-amylase and a thermolysin suitable for use in the liquefaction step of the method of the invention.

The enzyme composition of the present invention comprises:

i) Bacillus species alpha-amylase, or a variant thereof;

ii) a strong fireball protease;

In a preferred embodiment, the enzyme composition of the invention comprises glucoamylase and the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase: micrograms of glucoamylase).

In one embodiment, the alpha-amylase in the enzyme composition of the invention is a bacterial or fungal alpha-amylase.

In one embodiment, the alpha-amylase is derived from a Bacillus, e.g., bacillus stearothermophilus strain, particularly a variant of Bacillus stearothermophilus alpha-amylase, e.g., one of SEQ ID NO:3 or SEQ ID NO:1 as set forth herein in WO 99/019467.

In one embodiment, the bacillus stearothermophilus alpha-amylase or variant thereof is truncated, preferably truncated to have about 491 amino acids, e.g., from 480 to 495 amino acids.

In one embodiment, the Bacillus stearothermophilus alpha-amylase has a double deletion (preferably at position I181 +G182), and optionally has an N193F substitution, or a double deletion of R179 and G180 (numbered with SEQ ID NO: 1).

In one embodiment, the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ Having a T1/2 (min) of at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as between 10-70, such as between 15-70, such as between 20-70, such as between 25-70, such as between 30-70, such as between 40-70, such as between 50-70, such as between 60-70.

In one embodiment, the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants having the following mutations:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-I181 +g182 +n193F +v59A Q r+e129V +k177L +r179E +q254S +m284V; and

-I181 + g182 + N193F + E129V + K177L + R179E + K220P + N224L + s24q + Q254S (numbered using SEQ ID NO:1 herein).

In one embodiment, the alpha-amylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 1 herein.

In another embodiment, the alpha-amylase is a Bacillus licheniformis alpha-amylase, or a variant thereof.

In one embodiment, the Bacillus licheniformis alpha-amylase is one of the SEQ ID NOs shown herein as 21.

In one embodiment, the alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% identity to the mature portion of the polypeptide of SEQ ID NO. 21 herein.

In one embodiment, the enzyme composition comprises bacillus licheniformis alpha-amylase and a thermolysin.

In one embodiment, the enzyme composition further comprises a glucoamylase.

In one embodiment, the Pyrococcus furiosus is one of the SEQ ID NOS: 13 shown herein.

In one embodiment, the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

In one embodiment, the enzyme composition further comprises the glucoamylase shown in SEQ ID NO. 14, or a variant thereof.

In one embodiment, the glucoamylase is determined to have a thermostability of at least 20%, e.g., at least 30%, preferably at least 35%, at 85 ℃, pH 5.3 as disclosed in example 2 herein or in example 8 in WO 2011/127802.

In one embodiment, the glucoamylase is derived from a Penicillium strain, in particular a Penicillium oxalate strain as disclosed in SEQ ID NO. 2 of WO 2011/127802 or SEQ ID NO. 9 or 14 herein.

In one embodiment, the glucoamylase is a variant with a K79V substitution (numbered using the mature sequence shown in SEQ ID NO: 14) disclosed as SEQ ID NO:2 in WO 2011/127802 or as in SEQ ID NO:14 herein, e.g., the variant disclosed in WO 2013/053801.

T65A; or (b)

Q327F; or (b)

E501V; or (b)

Y504T; or (b)

Y504; or (b)

t65a+q327F; or (b)

t65a+e501V; or (b)

t65a+y504T; or (b)

T65a+y504; or (b)

Q327F+E501V; or (b)

Q327F+Y504T; or (b)

Q327 f+y504; or (b)

E501V+Y504T; or (b)

E501 v+y504; or (b)

T65a+q327f+e501V; or (b)

T65a+q327f+y504T; or (b)

T65a+e501v+y504T; or (b)

Q327F+E501V+Y504T; or (b)

T65a+q327 f+y504; or (b)

T65a+e501 v+y504; or (b)

Q327f+e501 v+y504; or (b)

T65a+q327f+e501v+y504T; or (b)

T65A+Q327F+E501V+Y504*；

E501V+Y504T; or (b)

t65a+k161S; or (b)

t65a+q405T; or (b)

t65a+q327W; or (b)

t65a+q327F; or (b)

t65a+q327Y; or (b)

p11f+t65a+q327F; or (b)

R1k+d3w+k5q+g7v+n8s+t10k+p1s+t65a+q 327F; or (b)

p2n+p4s+p11f+t65a+q327F; or (b)

p11f+d26c+k33c+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

R1e+d3n+p4g+g6r+g7a+n8a+t10d+p11d+t65a+q327F; or (b)

p11f+t65a+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P11f+t65a+q327w+e501v+y504T; or (b)

T65a+q327f+e501v+y504T; or (b)

t65a+s105p+q327W; or (b)

t65a+s105p+q327F; or (b)

T65a+q327w+s364P; or (b)

T65a+q327f+s364P; or (b)

t65a+s103n+q327F; or (b)

P2n+p4s+p11f+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447S; or (b)

P2n+p4s+p11f+t65a+i172v+q327F; or (b)

P2n+p4s+p11f+t65a+q327 f+n502; or (b)

P2n+p4s+p11f+t65a+q327f+n502t+p563s+k571E; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+n564d+k571S; or (b)

P2n+p4s+p11f+t65a+q327f+s377t; or (b)

P2n+p4s+p11f+t65a+v325t+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+i172v+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s377t+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+i375a+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k21a+k217d+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

P2n+p4s+t10d+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+f12y+t65a+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t568N; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+k524t+g526A; or (b)

P2n+p4s+p11f+k34y+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+f80+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k12s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+n502 t+y504; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+k9a+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9g+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9i+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9l+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+l72v+q327f+e501v+y504T; or (b)

S255n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+e7n+v79 k+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+g220n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+y245n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q257n+q7f+e501 v+y504T; or (b)

P2n+p4s+p11f+t65a+d279n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s359n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+d370n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460t+p468t+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+t463n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s465n+e501v+y504T; or (b)

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T。

In one embodiment, the glucoamylase is a penicillium oxalicum glucoamylase having a K79V substitution (numbered using SEQ ID NO: 14) and further having one of the following substitutions:

-P11F+T65A+Q327F

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

In one embodiment, the composition further comprises pullulanase.

In one embodiment, the pullulanase is a GH57 family pullulanase, wherein the pullulanase preferably comprises an X47 domain as disclosed in WO 2011/087836.

In one embodiment, the pullulanase is derived from strains from the genus thermophilic, including thermophilic and hydrothermal cocci, or hybrids thereof.

In one embodiment, the pullulanase is a Thermococcus bifidus pullulanase truncated at position X4 or a Thermococcus bifidus/Thermococcus hybrid having a truncation position X4, disclosed in WO 2011/087836 or shown in SEQ ID NO. 12 herein.

In one embodiment, the enzyme composition comprises:

-bacillus stearothermophilus alpha-amylase, or a variant thereof;

-a strong fireball protease; and

the enzyme Penicillium oxalicum glucoamylase,

In one embodiment, the enzyme composition of the invention comprises:

-a strong fireball protease; and

the enzyme Penicillium oxalicum glucoamylase,

In one embodiment, the enzyme composition comprises:

-an alpha-amylase derived from bacillus stearothermophilus having double deletions of I181+ G182 and substitutions of N193F; and additionally one of the following substitution sets:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

-a strong fireball protease; and

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbering using SEQ ID NO: 14).

In one embodiment, the ratio between alpha-amylase and protease is in the range of between 1:1.2 and 1:10, for example about 1:1.4 (micrograms of alpha-amylase: micrograms of protease).

In one embodiment, the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase: micrograms of glucoamylase).

The invention described and claimed herein is not intended to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

Materials and methods

Materials:

alpha-amylase A (AAA): bacillus stearothermophilus alpha-amylase having a mutation of I181 + G182 + N193F truncated to 491 amino acids (SEQ ID NO: 1)

Alpha-amylase 1407 (AA 1407):bacillus stearothermophilus alpha-amylase having the mutation I181 + G182 + N193F + V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S truncated to 491 amino acids (SEQ ID NO: 1)

Alpha-amylase 369 (AA 369): bacillus stearothermophilus alpha-amylase having the mutation I181 + G182 + N193F + V59A + Q89R + E129V + K177L + R179E + Q254S + M284V truncated to 491 amino acids (SEQ ID NO: 1).

Protease Pfu:a protease derived from Pyrococcus furiosus is shown in SEQ ID NO. 13 herein.

Glucoamylase Po: mature parts of the penicillium oxalicum glucoamylase disclosed as SEQ ID NO. 2 and shown herein in SEQ ID NO. 14 in WO 2011/127802.

Glucoamylase PoPE001:a variant of the penicillium oxalicum glucoamylase having the following mutations: K79V (encoded using SEQ ID NO: 14).

Glucoamylase Po 498(GA 498): a variant of the penicillium oxalicum glucoamylase having the following mutations: k79 V+P2N+P4S+P1F+T65A+Q327F (numbered using SEQ ID NO: 14).

Glucoamylase a:a blend comprising: the Emerson basket glucoamylase disclosed as SEQ ID NO 34 in WO 99/28448, the Thrombin annuloplast glucoamylase disclosed as SEQ ID NO 2 in WO 06/69289, and the Rhizomucor miehei alpha amylase (AGU: AGU: FAU-F with an activity ratio of about 20:5:1) having an Aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD) in SEQ ID NO 16 disclosed herein.

Glucoamylase U:a blend comprising: the Emerson basket glucoamylase disclosed as SEQ ID NO. 34 in WO 99/28448, the Thrombin annuloplast glucoamylase disclosed as SEQ ID NO. 2 in WO 06/69289, and the Rhizomucor miehei alpha-amylase having an Aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD) disclosed in SEQ ID NO. 16 (AGU: AGU: FAU-F with an activity ratio of about 65:15:1).

Protease X:metalloproteases from Thermoascus aureofaciens CGMCC No.0670 disclosed as amino acids 1-177 in SEQ ID NO. 3 and amino acids 1-177 in SEQ ID NO. 2 of WO 2003/048353

Yeast:

ETHANOL RED from Fuse Tex Co (Fermentis) of America ^TM

Method

Consistency: the relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For the purposes of the present invention, the degree of identity between two amino acid sequences and the degree of identity between two nucleotide sequences can be determined by the procedure "alignment", which is a Nidelman-Wunsch alignment (i.e., an overall alignment). This procedure was used to align polypeptides and nucleotide sequences. Polypeptide alignment was performed using the default scoring matrix BLOSUM50 and nucleotide alignment was performed using the default identity matrix. The penalty for the first residue of a gap is-12 for a polypeptide and-16 for a nucleotide. The penalty for a gap additional residue is-2 for the polypeptide and-4 for the nucleotide.

"alignment" is part of the FASTA package version v20u6 (see pearson (W.R. Pearson) and lipman (D.J. Lipman) (1988), improved tools for biological sequence analysis ("Improved Tools for Biological Sequence Analysis"), PNAS 85:2444-2448, and pearson (1990) rapid and sensitive sequence comparison using FASTP and FASTA ("Rapid and Sensitive Sequence Comparison with FASTP and FASTA") enzymology methods (Methods in Enzymology) 183:63-98). FASTA protein alignment was performed using the Smith-whatman algorithm (Smith-Waterman algorithm) with no limitation on gap size (see Smith-whatman algorithm ("Smith-Waterman algorithm"), smith (t.f.smith) and whatman (m.s.waterman) (1981) journal of molecular and biology (j.mol.biol.) 147:195-197).

Protease assay

AZCL-Casein assay

A0.2% solution of the blue substrate AZCL-casein was suspended in a BORAX/NaH at pH 9 with stirring ₂ PO ₄ In a buffer. The solution was dispersed on a microtiter plate (100. Mu.l per well) with stirring, and 30. Mu.l was addedThe enzyme samples were raised and the plates incubated in a Ai Bende hot mixer (Eppendorf Thermomixer) for 30 minutes at 45℃and 600 rpm. A denatured enzyme sample (boiled at 100 ℃ for 20 min) was used as a blank. After incubation the reaction was terminated by transferring the microtiter plate onto ice and the coloured solution was separated from the solids by centrifugation at 3000rpm for 5 minutes at 4 ℃. 60 microliters of the supernatant was transferred to a microtiter plate and absorbance at 595nm was measured using a buret microplate reader (BioRad Microplate Reader).

pNA assay

50 microliter of protease-containing sample was added to the microtiter plate and the sample was purified by adding 100 microliter of 1mM pNA substrate (5 mg dissolved in 100 microliter DMSO and further with Borax/NaH at pH 9.0 ₂ PO ₄ Dilution of buffer to 10 mL) starts the assay. OD (optical density) ₄₀₅ The increase at room temperature was monitored as a measure of protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in glucoamylase units (AGU).

Novo glucoamylase units (AGU) are defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under the following standard conditions: 37 ℃, pH 4.3, substrate: maltose 23.2mM, buffer: acetate 0.1M, reaction time 5 min.

An automated analyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent, thereby converting any alpha-D-glucose present to beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the above-mentioned reaction to form NADH, which is measured as a measure of the original glucose concentration at 340nm using a photometer.

AMG incubation:
		a substrate:	maltose 23.2mM
Buffer solution:	acetate 0.1M
		pH：	4.30±0.05
Incubation temperature:	37℃±1
		reaction time:	for 5 minutes
Enzyme working range:	0.5AGU/mL to 4.0AGU/mL

Color reaction:
		GlucDH：	430U/L
mutarotase:	9U/L
		NAD：	0.21mM
buffer solution:	phosphate 0.12M;0.15M NaCl
		pH：	7.60±0.05
Incubation temperature:	37℃±1
		reaction time:	for 5 minutes
Wavelength:	340nm

file @ describing this analysis method in more detailEB-SM-0131.02/01) Acquisition may be requested from Novozymes A/S, denmark, which is incorporated herein by reference.

Acid alpha-amylase activity

When used in accordance with the present invention, the activity of an acid alpha-amylase may be measured in AFAU (acid fungal alpha-amylase units) or FAU-F.

Acid alpha-amylase activity (AFAU)

The acid alpha-amylase activity can be measured in AFAU (acid fungal alpha-amylase units), which is determined relative to an enzyme standard. 1AFAU is defined as the amount of enzyme that degrades 5.260mg starch dry matter per hour under the standard conditions described below.

Acid alpha-amylases are endo-alpha-amylases (1, 4-alpha-D-glucan-glucohydrolase, e.c. 3.2.1.1) that hydrolyze alpha-1, 4-glycosidic bonds in the interior region of starch molecules to form dextrins and oligosaccharides with different chain lengths. The intensity of the color formed with iodine is proportional to the concentration of starch. Enzyme activity was measured as a decrease in starch concentration under the indicated assay conditions using reverse colorimetry.

Blue/violet t=23 seconds decolorization

Standard conditions/reaction conditions:

folder describing this analysis method in more detailEB-SM-0259.02/01Available from novelian corporation of denmark, incorporated herein by reference.

Determination of FAU-F

Measurement of FAU-F relative to an enzyme standard with declarative StrengthFungal alpha-amylase units(Fencryptal (Fungamyl))。

A folder (EB-SM-0216.02) describing this standard method in more detail is available from NoveXin corporation of Denmark, which folder is hereby incorporated by reference.

Alpha-amylase Activity (KNU)

The alpha-amylase activity may be measured using potato starch as a substrate. This method is based on potato starch modified by enzymatic decomposition, and the reaction is followed by mixing a sample of starch/enzyme solution with an iodine solution. Initially a reddish blue colour was formed but the blue colour weakens during starch decomposition and then gradually turns reddish brown, which was compared with a coloured glass standard.

A Kilo Novo alpha-amylase unit (KNU) is defined as in the standard barUnder-part (i.e., at 37 ℃ C. +/-0.05;0.0003M Ca) ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the And pH 5.6) an enzyme amount that dextrinizes 5260mg of starch dry matter merck soluble starch (Merck Amylum solubile).

The folder EB-SM-0009.02/01 describing this analysis in more detail is available from novelian company of denmark, which folder is hereby incorporated by reference.

Determination of pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN was measured relative to a novelin pullulanase standard. One pullulanase unit (NPUN) was defined as the amount of enzyme that released 1 micromole of glucose per minute under standard conditions (0.7% red pullulan (Megazyme), pH 5, 40 ℃,20 minutes).

1mL of the diluted sample or standard was incubated at 40℃for 2 minutes. 0.5mL of 2% red pullulan, 0.5M KCl, 50mM citric acid, pH 5 was added and mixed. The tubes were incubated at 40℃for 20 min and terminated by the addition of 2.5ml 80% ethanol. The tubes were allowed to stand at room temperature for 10-60 minutes, followed by centrifugation at 4000rpm for 10 minutes. The OD of the supernatant was then measured at 510nm and activity calculated using a standard curve.

The invention will be described in more detail in the following examples, which are provided to illustrate the invention and are in no way intended to limit the scope of the invention as claimed. All references cited herein are specifically incorporated by reference for the description set forth herein.

Examples

Example 1

Stability of alpha-amylase variants

By truncating a reference alpha-amylase (Bacillus stearothermophilus alpha-amylase truncated to 491 amino acids with mutation I181 + G182 + N193F (numbering as SEQ ID NO: 1)) and alpha-amylase variants thereof with 0.12mM CaCl at pH 4.5 and 5.5 and at temperatures of 75℃and 85 DEG ₂ Incubation is carried out, followed by use ofSubstrate [ ]The stability of the reference alpha-amylase and variants was measured by residual activity measurements using a super amylase assay kit (Ultra Amylase assay kit), E33651, molecular Probes).

Purified enzyme samples were incubated in enzyme dilution buffer (10 mM acetate, 0.01% Triton X100, 0.12mM CaCl ₂ pH 5.0) to a working concentration of 0.5 and 1 or 5 and 10ppm (micrograms/milliliter). Twenty microliters of enzyme sample was transferred to 48-well PCR MTP and 180 microliters of stability buffer (150 mM acetate, 150mM MES, 0.01% Triton X100, 0.12mM CaCl ₂ pH 4.5 or 5.5) was added to each well and mixed. The assay was repeated twice using two concentrations of enzyme. Before incubation at 75 ℃ or 85 ℃, 20 microliters were removed and stored on ice as a control sample. Incubation was performed in a PCR instrument (at 75 ℃ and 85 ℃). After incubation, the samples were incubated in residual activity buffer (100 mM acetate, 0.01% Triton X100, 0.12mM CaCl ₂ pH 5.5) to 15ng/ml and 25. Mu.l of diluting enzyme were transferred to black 384-MTP. Residual activity was measured using the EnzChek substrate by adding 25 microliters of substrate solution (100 micrograms/ml) to each well. Fluorescence was measured every minute for 15 minutes using an excitation filter at 485-P nm and an emission filter at 555nm (the fluorescence reader is polar, BMG). For each setting, residual activity was normalized to the control sample.

Assuming logarithmic decay, the half-life time (T1/2 (min)) is calculated using the equation T1/2 (min) =t (min) ×ln (0.5)/LN (% RA/100), where T is the assay incubation time in minutes and% RA is the% residual activity determined in the assay.

Using this assay setup, half-life times were determined for reference alpha-amylase and variants thereof, as shown in table 1.

TABLE 1

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ND does not detect

These results demonstrate that the alpha-amylase variants have significantly higher half-lives and stabilities than the reference alpha-amylase.

Example 2

Characterization of Penicillium oxalicum glucoamylase

The penicillium oxalicum glucoamylase is disclosed in SEQ ID NO. 9 herein.

A substrate. A substrate: reaction buffer containing 1% soluble starch (Sigma) S-9765 in deionized water: 0.1M acetate buffer, pH 5.3

Glucose concentration assay kit: and a light glucose assay kit (Labassay glucose, and light Co., ltd., catalog number 298-65701).

Reaction conditions. 20. Mu.l of soluble starch were mixed with 50. Mu.l of acetate buffer (pH 5.3). 30. Mu.l of enzyme solution (50. Mu.g enzyme protein/ml) was added to a final volume of 100. Mu.l, followed by incubation at 37℃for 15 minutes.

Glucose concentration was determined by means of a kit of parts (Wako kits).

All these works were performed in parallel.

Optimum temperature. In order to evaluate the optimum temperature of the penicillium oxalicum glucoamylase, the "reaction conditions" described above were measured at 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 85 ℃, 90 ℃ and 95 ℃. The results are shown in Table 2.

TABLE 2 optimum temperature

Temperature (. Degree. C.)	20	30	40	50	60	70	80	85	90	95
											Relative Activity (%)	63.6	71.7	86.4	99.4	94.6	100.0	92.9	92.5	82.7	82.8

From these results, it is known that the optimum temperature for penicillium oxalicum glucoamylase under the given conditions is between 50 ℃ and 70 ℃ and that the glucoamylase maintains more than 80% activity at 95 ℃.

Thermal stability. To evaluate the thermal stability of the penicillium oxalicum glucoamylase, the reaction condition assay was modified, wherein the enzyme solution and acetic acid buffer were pre-incubated at 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ and 95 ℃ for 15min. After incubation, 20 μl of starch was added to the solution and the assay was performed as described above.

The results are shown in Table 3.

TABLE 3 thermal stability

Temperature (. Degree. C.)	20	30	40	50	60	70	80	85	90	95
											Relative Activity (%)	91.0	92.9	88.1	100.0	96.9	86.0	34.8	36.0	34.2	34.8

From these results it is known that penicillium oxalicum glucoamylase is stable up to 70 ℃ after 15min pre-incubation, as it maintains more than 80% activity.

pH optimum. To evaluate the pH optimum of the penicillium oxalicum glucoamylase, the above reaction condition measurements were performed at the following pH: pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0.7.0, 8.0, 9.0, 10.0 and 11.0. Instead of the acetate buffers described in the reaction condition assay, the following buffers were used: 100mM succinic acid, HEPES, CHES, CAPSO, 1mM CaCl ₂ 150mM KCl, 0.01% Triton X-100, pH adjusted to 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl or NaOH.

The results are shown in Table 4.

TABLE 4pH optimum

From these results, it is clear that the Penicillium oxalicum glucoamylase has the highest activity at pH 5.0 under the given conditions. The penicillium oxalicum glucoamylase is active over a wide pH range, as it maintains over 50% activity from pH 2 to 7.

pH stability. To evaluate the thermostability of the penicillium oxalicum glucoamylase, the reaction condition assay was modified, wherein the enzyme solution (50 micrograms/mL) was pre-incubated for 20 hours in a buffer with pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 using the buffer at the pH optimum described above. After pre-incubation, 20 microliters of soluble starch was added to the solution to a final volume of 100 microliters and the assay was performed as described above.

The results are shown in Table 5.

TABLE 5pH stability

From these results, it is known that penicillium oxalicum glucoamylase is stable from pH 3 to pH7 after 20 hours of pre-incubation and it decreases activity at pH 8.

Example 3

Thermal stability of Penicillium oxalicum glucoamylase variant (PoAMG) -by Differential Scanning Calorimetry (DSC) Analysis

Site-specific penicillium oxalicum glucoamylase (PoAMG) variants with substitutions and/or deletions at specific positions were constructed essentially as described in example 3 and purified as described in example 4 in WO 2013/053801 (hereby incorporated by reference).

The thermostability of the purified glucoamylase Po PE001 (SEQ ID NO:14 with K79V) derived variant was determined by Differential Scanning Calorimetry (DSC) at pH 4.0 or 4.8 (50 mM sodium acetate) using a VP-capillary differential scanning calorimeter (Michael Inc. of Piscataway, N.J.). The thermal denaturation temperature Td (. Degree.C.) was taken as the top of the denaturation peak (the major endothermic peak) in the thermal analysis plot (Cp vs T) obtained after heating the enzyme solution in the selected buffer (50 mM sodium acetate, pH 4.0 or 4.8) at a constant programmed heating rate of 200K/hr.

Samples and reference solutions (about 0.3 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10 ℃ and thermally pre-equilibrated at 20 ℃ for 10 minutes followed by DSC scans from 20 ℃ to 110 ℃. The denaturation temperature was determined with an accuracy of about +/-1 ℃.

Isolated variants and DSC data are disclosed in table 6 below.

Table 6.

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Example 4

Use of high dose protease Pfu for oil extraction and ethanol

Liquefying: nine slurries of whole corn flour, reflux and tap water were prepared to a total weight of 150g targeting 32.50% Dry Solids (DS); reflux was blended at 30% reflux weight/slurry weight. The slurry pH was 5.0 and no additional adjustments were made prior to using the following treatments:

3 mash are controls, meaning that they only accept alpha-amylase 369 (AA 369) during liquefaction and will be baseline. When applied, AA369 was applied in a fixed dose of 2.1 μg/gDS for all cases.

2 mash were treated with AA369 and 1.5. Mu.g/g DS protease Pfu.

2 mash were treated with AA369 and 3. Mu.g/g DS protease Pfu.

2 mash were treated with AA369 and 5. Mu.g/g DS protease Pfu.

Water and enzyme were added to each tank and then each tank was sealed and thoroughly mixed prior to loading into the Labomat. All samples were incubated in Labomat set to the following conditions: 5 ℃/min. Gradual, 15 minutes to 80 ℃, hold for 1min, gradual at 1 ℃/min to 85 ℃ and hold 103min,40rpm turn left for 30 seconds and right for 30 seconds. Once liquefaction was completed, all tanks were cooled in an ice bath for about 20 minutes before fermentation was performed.

Simultaneous Saccharification and Fermentation (SSF): penicillin was added to each mash to a final concentration of 3ppm and adjusted to pH 5.0 with 40% sulfuric acid or 45% potassium hydroxide as needed. A portion of this mash is then transferred to a test tube and represents a "urea free" fermentation, or what is considered to be a nitrogen limited fermentation. Once the "urea free" mash is treated, urea is added to the remaining mash up to a final concentration of 200ppm and transferred to a test tube for fermentation. All test tubes were drilled with a 1/64' drill bit to allow CO ₂ Releasing. Furthermore, the equivalent solids are maintained by the addition of water as required to ensure that the urea and urea-free mash contain equivalent solids in all treatments. Fermentation was started by adding glucoamylase A (0.60 AGU/gDS), water, and rehydrated yeast. By adding 5.5g of ETHANOL RED from Fuse Tex Co., fermentis ^TM Mix into 100mL of tap water at 32 ℃ for at least 15 minutes and give 100 μl per tube for yeast rehydration.

And (3) distilling: bu Qi (Buchi) parallel evaporator evaporation system was used for all distillations. The device distills 12 samples simultaneously. The parameters used are shown in table 7. The tubes were weighed after distillation and the weight loss during distillation was replaced with DI water. The tubes were again weighed after the water addition. Three separate distillations were performed for this test, which included one control per run.

TABLE 7 distillation parameters for corn oil determination

Oil extraction: hexane was added to each sample at a dose of 0.125mL hexane/1 g starting material. Each tube was covered in a sealing film (Dura-seal) to prevent sample leakage and thoroughly mixed. In an Avanti JE series centrifuge with JS-5.3 rotor, the tube was centrifuged at 3,000Xg for 10 minutes. After centrifugation, the oil/hexane layer (supernatant) was removed using a positive displacement pipette, transferred to a pre-weighed 5mL flip top tube, and re-weighed. Sample density was measured using a ruff study analysis densitometer. The density of the supernatant was then calculated using the standard curve equation to find the% oil in the supernatant. From this value the total% of oil in the starting material was derived.

HPLC analysis: HPLC analysis used Agilent 1100/1200 combined with a Berle company (Bio-Rad) HPX-87H ion exclusion column (300 mm x 7.8 mm) and a Berle company cation H guard column. The mobile phase was 0.005M sulfuric acid at a flow rate of 0.6ml/min and the treated sample, and the column and RI detector temperatures were 65℃and 55℃respectively. After 54 hours, samples were taken by fermentation through 3 tubes sacrificed per treatment. Each tube was prepared by mixing with 50. Mu.L of 40% v/v H ₂ SO ₄ Inactivation, vortexing, centrifugation at 1460×g for 10 min, and filtration through 0.45 μm whatman PP filter (Whatman PP filter). Samples were stored at 4 ℃ before and during HPLC analysis. The method uses calibration standards for dp4+, DP3, DP2, glucose, fructose, acetic acid, lactic acid, glycerol, and ethanol (% w/v) to quantify the analytes. Quantification was performed using four-point calibration (including source).

Results (corn oil extraction): the terminology used in the examples: for corn oil extraction, there were three separate controls (controls A, B and C) because one control was treated for each distillation run. However, each control was treated during liquefaction and fermentation: there is no protease during the fermentation process in which protease X is added to the fermentation. Extraction of corn oil showed that the lowest dose added to liquefied protease Pfu matched the dose of fermented protease X (0.025% w/w) (figure 1). Furthermore, increasing proteases Pfu to 3 and 5 μg/gDS over the oil recovered with protease X alone, and there was no additional benefit in combining both protease Pfu and protease X at this dose, indicated little synergy between the two proteases (fig. 2 and 3).

Ethanol: performance of different proteases on limited doses of exogenous nitrogen addition

The performance of different proteases with limited doses of exogenous nitrogen was also tested. It was investigated how substrates produced from these proteases affect fermentation rate, carbohydrate consumption and glycerol formation.

Treatment of the fermentation with proteinase X and without supplementation with nitrogen from urea did not result in drying, which was provided at 54 hour mark for all doses (FIG. 4). Furthermore, increasing the protease Pfu to 3 or 5 μg/gDS from 1.5 μg/gDS resulted in a significant increase (% w/v) in final ethanol concentration (fig. 4 and 5). These different protease treatments also demonstrated an effect on the fermentation rate when the 24 hour ethanol concentration was highest in the case of the highest treatment with protease Pfu.

A similar trend was observed when 200ppm urea was incorporated into the fermentation, but by providing the lowest residual glucose and producing the highest final ethanol, the superior protease Pfu activity in the liquefaction process was maintained relative to protease X. Thus, by supporting faster fermentation over the first 24 hours, combined with a statistically higher final ethanol concentration (%w/v), the lowest dose of protease Pfu (1.5. Mu.g/gDS) performed better than protease X (FIGS. 6 and 7). Overall, although the two highest doses were statistically equivalent in this example, the more protease Pfu at the 54 hour mark resulted in more ethanol production. All fermentations reached a dry state at the 54 hour mark. Higher doses of protease Pfu also reduced glycerol formation during fermentation, and this shift in metabolism was part of the reason for the observed increase in ethanol (FIG. 8-%w/v).

Conclusion(s)

Oil extraction:

1.5. Mu.g/gDS protease Pfu action combined with 2.1. Mu.g EP/gDS alpha-amylase 369 in conventional corn-based liquefaction (85 ℃, pH 5.0,2 hours) work matched the increase in oil extracted from fermentation treatment with protease X (5. Mu.g/gDS).

An even higher protease Pfu dose of 3 μg/gDS resulted in about 5% more oil than protease X alone, and similar results were seen with 5 μg/gDS dose.

There is no obvious synergy in oil recovery when the protease Pfu is combined with protease X and there appears to be no improvement in running the protease during liquefaction compared to fermentation.

Ethanol yield: the protease Pfu shows superior properties over protease X

Urea free:

the o 24 hour data shows that protease Pfu (1.5 μg/gDS) provides a more efficient fermentation than protease X by having lower residual glucose and higher ethanol concentration, with an increase to 3 or 5 μg/gDS resulting in even more ethanol production.

The o 54 hour data shows that protease Pfu (1.5. Mu.g/gDS) performs better than protease X by providing low residual glucose and higher ethanol concentrations. Drying was achieved with all doses of protease Pfu fermentation, however protease X was completed only in excess of 1% w/v.

The glycerol concentration of o-protease Pfu (5. Mu.g/gDS) was 10% lower than that of protease X.

200ppm urea:

the o 24 hour data shows that protease Pfu (1.5. Mu.g/gDS) performs better than protease X by having a low residual glucose and higher ethanol concentration at 200ppm urea. In general, although the two highest doses (3 and 5. Mu.g/gDS) are very similar, the more protease Pfu by this point results in more ethanol.

o 54 h HPLC showed that protease Pfu (1.5. Mu.g/gDS) performed better than protease X by providing the lowest residual glucose while producing the highest ethanol. All fermentations were dry. Even the lowest dose of protease Pfu (1.5. Mu.g/gDS) produced 1% more ethanol than protease X. Although the proteases Pfu of 3 and 5. Mu.g/gDS are statistically equivalent on the final ethanol, they are both higher than the lowest protease Pfu dose.

The glycerol concentration of o-protease Pfu (5. Mu.g/gDS) was about 9% lower than that of protease X. The protease Pfu provides the optimal final ethanol concentration while also providing the lowest final glycerol concentration.

Example 5

Use of high protease Pfu doses in liquefaction process in ethanol production process

Liquefying: thirteen slurries of whole corn flour and tap water were prepared to a total weight of 125g targeting 32.50% Dry Solids (DS); reflux was blended at 30% reflux weight/slurry weight. The initial slurry pH was about 6.0 and was adjusted to 5.0 with 45% w/v potassium hydroxide or 40% v/v sulfuric acid. A fixed dose of alpha-amylase 1407 (1.73 μg EP/gDS) was applied to all slurries and combined with the following protease Pfu to assess the effect of high protease treatment during liquefaction:

control: alpha-amylase only

Alpha-amylase 1407+0.0355. Mu.g/gDS protease Pfu

Alpha-amylase 1407+0.25. Mu.g/gDS protease Pfu

Alpha-amylase 1047+0.5. Mu.g/gDS protease Pfu

Alpha-amylase 1407+1. Mu.g/gDS protease Pfu

Alpha-amylase 1407+10. Mu.g/gDS protease Pfu

Alpha-amylase 1407+50. Mu.g/gDS protease Pfu

Simultaneous Saccharification and Fermentation (SSF): penicillin was added to each mash to a final concentration of 3ppm and the pH was adjusted to 5.0. Next, a portion of the mash was transferred into a test tube to represent a "urea free" fermentation and was considered nitrogen limited. Once the "urea free" mash is treated, urea is administered in the remaining mash up to a final concentration of 800ppm and is also transferred to a test tube. All test tubes were drilled with a 1/64' drill bit to allow CO ₂ Releasing. Furthermore, the equivalent solids are maintained by the addition of water as required to ensure that the urea and urea-free mash contain equivalent solids in all treatments. By adding glucoamylase U (0.50 AGU/gDS), water and rehydrationTo initiate fermentation. By mixing 5.5g of ETHANOL RED ^TM Mix into 100mL of tap water at 32 ℃ for at least 15 minutes and give 100 μl per tube for yeast rehydration.

Conclusion: comparison of the 54 hour ethanol concentration showed that more than 1 μg/gDS protease Pfu was required under nitrogen limited conditions to support fermentation to dryness (figures 9 and 10). Furthermore, the remaining glucose was 0.4% w/v for the 1 μg/gDS dose, whereas no remaining glucose was observed at the protease Pfu dose of 10 or 50 μg/gDS. The most significant reductions in glycerol were observed at the highest doses of protease Pfu, i.e. 10 and 50. Mu.g/gDS. These results indicate that as much as 10 to 50 μg/gDS of protease Pfu may be required in the liquefaction process to achieve optimal performance involving liquefaction and fermentation.

Summary paragraphs

The invention is defined in the claims and the accompanying description. For convenience, other aspects of the invention are presented herein by numbering paragraphs:

1. a method of recovering oil from a fermentation product production process, the method comprising the steps of:

-an alpha-amylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

2. The method of paragraph 1, wherein the oil is recovered during and/or after liquefying the starch-containing material.

3. The method of paragraph 1 wherein oil is recovered from the stillage.

4. The method of any of paragraphs 1-3, wherein the oil is recovered from thin stillage.

5. The method of any of paragraphs 1-4, wherein oil is recovered from the slurry.

6. The method of any of paragraphs 1-5, wherein 0.5-100 microgram of pyrococcus furiosus protease per gram DS, e.g. 1-50 microgram of pyrococcus furiosus protease per gram DS, e.g. 1-10 microgram of pyrococcus furiosus protease per gram DS, e.g. 1.5-5 microgram of pyrococcus furiosus protease per gram DS, e.g. about or greater than 1.5 microgram of pyrococcus furiosus protease per gram DS, is present and/or added in liquefaction step a).

7. The method of any of paragraphs 1-6, wherein 2-100 micrograms of the pneumococcal protease per gram DS, e.g. 2.5-50 micrograms of the pneumococcal protease per gram DS, e.g. 2.5-10 micrograms of the pneumococcal protease per gram DS, e.g. 2.5-5 micrograms of the pneumococcal protease per gram DS, especially about 3 micrograms of the pneumococcal protease per gram DS, is present and/or added in liquefaction step a).

8. The method of any one of paragraphs 1-7, wherein the strong fireball protease is the mature sequence set forth in SEQ ID NO. 13 herein.

9. The method of any of paragraphs 1-8, wherein the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

10. The method of any of paragraphs 1-9, wherein in steps a) -c), no nitrogen compounds are present and/or added, e.g. during saccharification step b), fermentation step c), or Simultaneous Saccharification and Fermentation (SSF).

11. The method of any of paragraphs 1-10, wherein in steps a) -c), e.g. in saccharification step b) or fermentation step c) or Simultaneous Saccharification and Fermentation (SSF), 10-1,000ppm, e.g. 50-800ppm, e.g. 100-600ppm, e.g. 200-500ppm of nitrogen compound (preferably urea) is present and/or added.

12. The method of any of paragraphs 1-11, wherein the alpha-amylase is from a bacillus, e.g., a bacillus stearothermophilus strain, e.g., the sequence set forth in SEQ ID No. 1.

13. The method of any one of paragraphs 1-12, wherein the alpha-amylase is a bacillus stearothermophilus alpha-amylase shown in SEQ ID No. 1 herein, e.g., an alpha-amylase having at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity to SEQ ID No. 1 herein.

14. The method of paragraph 11, wherein the bacillus stearothermophilus alpha-amylase or variant thereof is truncated, preferably truncated to have about 491 amino acids, e.g., from 480 to 495 amino acids.

15. The method of any one of paragraphs 12-14, wherein the Bacillus stearothermophilus alpha-amylase has a double deletion at position I181+ G182 and optionally has an N193F substitution (numbering using SEQ ID NO: 1),

16. the method of any one of paragraphs 12-14, wherein the bacillus stearothermophilus alpha-amylase has a double deletion at position R179+ G180 and optionally has an N193F substitution (numbering using SEQ ID NO: 1).

17. The method of any one of paragraphs 12-16, wherein the bacillus stearothermophilus alpha-amylase has a substitution at position S242, preferably a S242Q substitution.

18. The method of any one of paragraphs 12-17, wherein the bacillus stearothermophilus alpha-amylase has a substitution at position E188, preferably an E188P substitution.

19. The method of any one of paragraphs 1-18, wherein the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ The lower part has a T1/2 (min) of at least 10.

20. The method of any one of paragraphs 1-19, wherein the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ Having a T1/2 (min) of at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as between 10-70, such as between 15-70, such as between 20-70, such as between 25-70, such as between 30-70, such as between 40-70, such as between 50-70, such as between 60-70.

21. The method of any one of paragraphs 1-20, wherein the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants having the following mutations in addition to I181 x + G182 x and optionally N193F:

/>

22. the method of any one of paragraphs 1-21, wherein the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

-i181+g182+n193 f+v59aq9r+e129 v+k177l+r179e+q254s+m284V; and

23. The method of any one of paragraphs 1-22, wherein the alpha-amylase variant has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID NO:1 herein.

24. The method of any one of paragraphs 1-23, wherein the alpha-amylase is a bacillus licheniformis alpha-amylase, or variant thereof.

25. The method of paragraph 24 wherein the Bacillus licheniformis alpha-amylase is one of the SEQ ID NOs as shown herein as 21.

26. The method of any one of paragraphs 1-25, wherein the alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% identity to the mature portion of the polypeptide of SEQ ID No. 21 herein.

27. The method of any of paragraphs 1-26, wherein the alpha-amylase may be present and/or added at a concentration of 0.1-100 micrograms/gram DS, e.g., 0.5-50 micrograms/gram DS, e.g., 1-25 micrograms/gram DS, e.g., 1-10 micrograms/gram DS, e.g., 2-5 micrograms/gram DS.

28. The method of any one of paragraphs 1-27, wherein during liquefaction, from 1-10 micrograms of the thermolysin enzyme and 1-10 micrograms of the bacillus stearothermophilus alpha-amylase are present and/or added.

29. The method of any one of paragraphs 1-28, wherein glucoamylase is present and/or added in liquefaction step a).

30. The method of paragraph 29, wherein the glucoamylase present and/or added during liquefaction has a thermostability of at least 20%, e.g., at least 30%, preferably at least 35% at 85 ℃, pH 5.3.

31. The method of paragraph 29 or 30, wherein the glucoamylase has a relative activity of at least 90%, preferably at least 95%, preferably at least 97% at a pH optimum of pH 5.0.

32. The method of any one of paragraphs 29-30, wherein the glucoamylase has a pH stability of at least 80%, at least 85%, at least 90% at pH 5.0.

33. The method of any of paragraphs 29-32, wherein the glucoamylase present and/or added during liquefaction step a) is derived from a penicillium strain, in particular a penicillium oxalate strain as disclosed in WO 2011/127802 as SEQ ID No. 2 or as SEQ ID No. 9 or 14 herein.

34. The method of paragraphs 29-33, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the mature polypeptide shown in SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 9 or 14 herein.

35. The method of any one of paragraphs 29-34, wherein the glucoamylase is a variant of the penicillium oxalicum glucoamylase of SEQ ID No. 2 shown in WO 2011/127802 having a K79V substitution (numbering using the mature sequence shown in SEQ ID No. 14), e.g., the variant disclosed in WO 2013/053801.

36. The method of any one of paragraphs 29-35, wherein the penicillium oxalicum glucoamylase has a K79V substitution (numbered using SEQ ID NO: 14) and further has one of:

T65A; or (b)

Q327F; or (b)

E501V; or (b)

Y504T; or (b)

Y504; or (b)

t65a+q327F; or (b)

t65a+e501V; or (b)

t65a+y504T; or (b)

T65a+y504; or (b)

Q327F+E501V; or (b)

Q327F+Y504T; or (b)

Q327 f+y504; or (b)

E501V+Y504T; or (b)

E501 v+y504; or (b)

T65a+q327f+e501V; or (b)

T65a+q327f+y504T; or (b)

T65a+e501v+y504T; or (b)

Q327F+E501V+Y504T; or (b)

T65a+q327 f+y504; or (b)

T65a+e501 v+y504; or (b)

Q327f+e501 v+y504; or (b)

T65a+q327f+e501v+y504T; or (b)

T65A+Q327F+E501V+Y504*；

E501V+Y504T; or (b)

t65a+k161S; or (b)

t65a+q405T; or (b)

t65a+q327W; or (b)

t65a+q327F; or (b)

t65a+q327Y; or (b)

p11f+t65a+q327F; or (b)

R1k+d3w+k5q+g7v+n8s+t10k+p1s+t65a+q 327F; or (b)

p2n+p4s+p11f+t65a+q327F; or (b)

p11f+d26c+k33c+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

R1e+d3n+p4g+g6r+g7a+n8a+t10d+p11d+t65a+q327F; or (b)

p11f+t65a+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P11f+t65a+q327w+e501v+y504T; or (b)

T65a+q327f+e501v+y504T; or (b)

t65a+s105p+q327W; or (b)

t65a+s105p+q327F; or (b)

T65a+q327w+s364P; or (b)

T65a+q327f+s364P; or (b)

t65a+s103n+q327F; or (b)

P2n+p4s+p11f+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447S; or (b)

P2n+p4s+p11f+t65a+i172v+q327F; or (b)

P2n+p4s+p11f+t65a+q327 f+n502; or (b)

P2n+p4s+p11f+t65a+q327f+n502t+p563s+k571E; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+n564d+k571S; or (b)

P2n+p4s+p11f+t65a+q327f+s377t; or (b)

P2n+p4s+p11f+t65a+v325t+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+i172v+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s377t+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+i375a+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k21a+k217d+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

P2n+p4s+t10d+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+f12y+t65a+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t568N; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+k524t+g526A; or (b)

P2n+p4s+p11f+k34y+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+f80+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k12s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+n502 t+y504; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+k9a+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9g+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9i+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9l+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+l72v+q327f+e501v+y504T; or (b)

S255n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+e7n+v79 k+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+g220n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+y245n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q257n+q7f+e501 v+y504T; or (b)

P2n+p4s+p11f+t65a+d279n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s359n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+d370n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460t+p468t+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+t463n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s465n+e501v+y504T; or (b)

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T。

37. The method of any one of paragraphs 29-36, wherein the glucoamylase present and/or added in liquefaction is a penicillium oxalicum glucoamylase having a K79V substitution (numbered with SEQ ID NO: 14) and further having one of:

-P11F+T65A+Q327F；

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

38. The method of any one of paragraphs 11-27, wherein the glucoamylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 14 herein.

39. The method of any one of paragraphs 1-38, further wherein glucoamylase is present and/or added during saccharification and/or fermentation.

40. The method of paragraph 39 wherein the glucoamylase present and/or added during saccharification and/or fermentation is of fungal origin, preferably from a strain of Aspergillus, preferably a strain of Aspergillus niger, aspergillus awamori, or Aspergillus oryzae; or a strain of Trichoderma, preferably a strain of Trichoderma reesei; or a strain of the genus panus, preferably panus emersonii; or a strain of midwifery; or a strain of the genus Myxophyllum, for example, myxophyllum palustris or Myxophyllum, or a strain of the genus Nicotiana.

41. The method of any of paragraphs 39-40, wherein the glucoamylase is derived from Emerson basket (e.g., one of SEQ ID NOS: 19 as set forth herein).

42. The method of any one of paragraphs 39-41, wherein the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 19 herein;

43. The method of any of paragraphs 39-42, wherein the glucoamylase is derived from Philippine (e.g., one of SEQ ID NOs: 15 as set forth herein).

44. The method of any one of paragraphs 39-43, wherein the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 15 herein;

45. The method of any of paragraphs 39-44, wherein the glucoamylase is derived from Philippine (e.g., one of SEQ ID NOs: 17 as set forth herein).

46. The method of any one of paragraphs 39-45, wherein the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 17 herein;

47. The method of any of paragraphs 39-46, wherein the glucoamylase is present and/or added in combination with an alpha-amylase during saccharification and/or fermentation.

48. The method of paragraph 47 wherein the alpha-amylase present and/or added during saccharification and/or fermentation is of fungal or bacterial origin.

49. The method of paragraph 47 or 48, wherein the alpha-amylase present and/or added in saccharification and/or fermentation is derived from a rhizomucor strain, preferably a strain of rhizomucor minium, such as one of SEQ ID nos. 3 shown in WO 2013/006756, such as a rhizomucor minium alpha-amylase hybrid having an aspergillus niger linker and a starch binding domain, such as one of SEQ ID nos. 16 shown herein, or a variant thereof.

50. The method of any one of paragraphs 47-49, wherein the alpha-amylase present and/or added in saccharification and/or fermentation is selected from the group consisting of:

(i) An alpha-amylase comprising the mature polypeptide of SEQ ID NO. 16 herein;

51. The method of any one of paragraphs 47-50, wherein the alpha-amylase is an alpha-amylase variant shown in SEQ ID No. 13 with at least one of the following substitutions or combinations: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; s123h+y141W; g20s+y141W; a76g+y141W; g128d+y141W; g128d+d143N; p219C+Y141W; n142d+d143N; y141w+k192R; y141w+d143N; y141w+n383R; y141w+p219c+a265C; y141 w+n517d+d143N; y141w+k192R V410A; g128d+y141w+d143N; y141w+d143n+p219C; y141w+d143n+k192R; g128d+d143n+k192R; y141w+d143 n+k168r+p219C; g217d+y141 w+d143n+k192R; or G128D+Y141W+D143N+K192R+P219C (numbered with SEQ ID NO: 16).

52. The method of any one of paragraphs 47-51, wherein the alpha-amylase is derived from rhizomucor parvum having an aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD), preferably as disclosed in SEQ ID No. 13 herein, preferably with one or more of the following substitutions: G128D, D N, preferably G128D+D143N (numbered using SEQ ID NO: 13).

53. The method of any one of paragraphs 47-52, wherein the alpha-amylase variant present and/or added in saccharification and/or fermentation has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 16 herein.

54. The method of any one of paragraphs 1-53, further wherein pullulanase is present and/or added during liquefaction and/or saccharification and/or fermentation.

55. The method of paragraph 54 wherein the pullulanase is a GH57 family pullulanase, wherein the pullulanase preferably comprises the X47 domain as disclosed in WO 2011/087836.

56. The method of paragraphs 54-55, wherein the pullulanase is derived from a strain from the genus thermophilic, including pyrococcus thermophilus and pyrococcus hydrothermal, or hybrids thereof.

57. The method of any of paragraphs 54-56, wherein the pullulanase is a hot water pyrococcus pullulanase truncated at position X4 or a hot water pyrococcus/thermophilic pyrococcus hybrid enzyme disclosed in WO 2011/087836 or shown in SEQ ID No. 12 herein having a truncation position X4.

58. The method of any of paragraphs 1-57, further comprising, prior to liquefying step a), the steps of:

ii) forming a slurry comprising the starch-containing material and water.

59. A method as claimed in any one of paragraphs 1-58, wherein at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material is suitable for passing through a screen having a #6 screen.

60. The method of any one of paragraphs 1-59, wherein the pH during liquefaction is between above 4.5-6.5, such as about 4.8, or between 5.0-6.2, such as 5.0-6.0, such as between 5.0-5.5, such as about 5.2, such as about 5.4, such as about 5.6, such as about 5.8.

61. The method of any one of paragraphs 1-60, wherein the temperature during liquefaction is above the initial gelatinization temperature, preferably in the range from 70-100 ℃, such as between 75-95 ℃, such as between 75-90 ℃, preferably between 80-90 ℃, especially about 85 ℃.

62. The method of any one of paragraphs 1-61, wherein a jet cooking step is performed prior to liquefaction in step a).

63. The method of paragraph 62, wherein the jet cooking is carried out at a temperature of 110 ℃ to 145 ℃, preferably 120 ℃ to 140 ℃, e.g. 125 ℃ to 135 ℃, preferably about 130 ℃ for about 1 to 15 minutes, preferably for about 3 to 10 minutes, especially about 5 minutes or so.

64. The method of any one of paragraphs 1-63, wherein saccharification and fermentation are conducted sequentially or simultaneously.

65. The method of any one of paragraphs 1-64, wherein saccharification is conducted at a temperature of from 20 ℃ -75 ℃, preferably from 40 ℃ -70 ℃, e.g., about 60 ℃, and at a pH of between 4 and 5.

66. The method of any one of paragraphs 1-65, wherein fermentation or Simultaneous Saccharification and Fermentation (SSF) is performed at a temperature of from 25 ℃ to 40 ℃, e.g., from 28 ℃ to 35 ℃, e.g., from 30 ℃ to 34 ℃, preferably about 32 ℃. In one embodiment, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.

67. The method of any one of paragraphs 1-66, wherein the fermentation product is recovered after fermentation, e.g., by distillation.

68. The method of any one of paragraphs 1-67, wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol, and/or industrial ethanol.

69. The method of any one of paragraphs 1-68, wherein the starch-containing starting material is whole grain.

70. The method of any one of paragraphs 1-69, wherein the starch-containing material is selected from the group consisting of: corn, wheat, barley, rye, milo, sago, tapioca, manioc, tapioca starch, sorghum, rice or potato.

71. The method of any one of paragraphs 1-70, wherein the fermenting microorganism is a yeast, preferably a saccharomyces strain, especially a saccharomyces cerevisiae strain.

72. The method of any one of paragraphs 1-71, wherein the alpha-amylase is a bacterial or fungal alpha-amylase.

73. The method of any one of paragraphs 1-72, wherein saccharification step b) and fermentation step c) are conducted simultaneously or sequentially.

74. The method of any one of paragraphs 1-73, wherein the temperature in step (a) is above the initial gelatinization temperature, e.g., at a temperature between 80-90 ℃, e.g., about 85 ℃.

75. The process of any one of paragraphs 1-74, further comprising a pre-saccharification step prior to saccharification step b), the pre-saccharification step being conducted at a temperature of between 30 ℃ and 65 ℃ for 40-90 minutes.

76. The method of any one of paragraphs 1-75, wherein saccharification is conducted at a temperature of from 20 ℃ -75 ℃, preferably from 40 ℃ -70 ℃, e.g., about 60 ℃, and at a pH between 4 and 5.

77. The method of any one of paragraphs 1-76, wherein fermentation step c) or Simultaneous Saccharification and Fermentation (SSF) (i.e., steps b) and c)) is conducted at a temperature of from 25 ℃ to 40 ℃, e.g., from 28 ℃ to 35 ℃, e.g., from 30 ℃ to 34 ℃, preferably about 32 ℃.

78. The method of any one of paragraphs 1-77, wherein the fermentation step c) or Simultaneous Saccharification and Fermentation (SSF) (i.e., steps b) and c)) is conducted for 6 to 120 hours, specifically 24 to 96 hours.

79. The method of any one of paragraphs 1-78, wherein the separation in step e) is by centrifugation, preferably a decanting centrifuge, filtration, preferably using a filter press, screw press, plate and frame press, gravity concentrator or dehydrator.

80. The method of any one of paragraphs 1-79, wherein the fermentation product is recovered by distillation.

81. The method of recovering oil of any one of paragraphs 1-80, comprising the steps of:

-the penicillium oxalate comprising a K79V substitution shown in SEQ ID No. 14;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

82. The method of recovering oil of any one of paragraphs 1-81, comprising the steps of:

bacillus stearothermophilus alpha-amylase comprising a double deletion at position I181+G182 and the following substitution N193F +V59A +Q9R+E129V +K177L +R179E +Q254S +M284V (numbering using SEQ ID NO: 1) truncated to 491 amino acids.

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

83. The method of any one of paragraphs 1-82, wherein the ratio between alpha-amylase and glucoamylase during liquefaction is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase/gram of DS: micrograms of glucoamylase/gram of DS).

84. The method of any of paragraphs 1-83, wherein the ratio between alpha-amylase and protease during liquefaction is between 1:1 and 1:25, e.g., in the range between 1:1.2 and 1:10, e.g., about 1:1.4 (micrograms of alpha-amylase/gram of DS: micrograms of protease/gram of DS).

85. A method for producing a fermentation product from starch-containing material, the method comprising the steps of:

-an alpha-amylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

86. The method of paragraph 85, wherein 2-100 micrograms/gram DS, e.g., 2.5-50 micrograms/gram DS, e.g., 2.5-10 micrograms/gram DS, e.g., 2.5-5 micrograms/gram DS, especially about 3 micrograms/gram DS, is a thermolysin.

87. The method of any of paragraphs 85 or 86, wherein the thermolysin is one of SEQ ID NOs 13 shown herein.

88. The method of any one of paragraphs 85-87, wherein the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

89. The method of any one of paragraphs 85-88, wherein in steps a) -c), no nitrogen compound is present and/or added, e.g., during saccharification step b), fermentation step c), or Simultaneous Saccharification and Fermentation (SSF).

90. The method of any one of paragraphs 85-89, wherein in steps a) -c), e.g. in saccharification step b) or fermentation step c) or Simultaneous Saccharification and Fermentation (SSF), 10-1,000ppm, e.g. 50-800ppm, e.g. 100-600ppm, e.g. 200-500ppm of nitrogen compound (preferably urea) is present and/or added.

91. The method of any one of paragraphs 85-90, wherein the alpha-amylase is from a bacillus, e.g., a bacillus stearothermophilus strain, particularly a variant of a bacillus stearothermophilus alpha-amylase, e.g., one of SEQ ID No. 3 or SEQ ID No. 1 herein shown, or a variant thereof, or a bacillus licheniformis strain, e.g., one of SEQ ID No. 21 herein shown, in WO 99/019467.

92. The method of paragraph 91 wherein the bacillus stearothermophilus alpha-amylase or variant thereof is truncated, preferably truncated to have about 491 amino acids, e.g., from 480 to 495 amino acids.

93. The method of any one of paragraphs 91 or 92, wherein the bacillus stearothermophilus alpha-amylase has a double deletion at position I181+ G182 and optionally has an N193F substitution, or R179 and G180 deletions (numbering using SEQ ID NO: 1).

94. The method of any one of paragraphs 85-93, wherein the bacillus stearothermophilus alpha-amylase has a substitution at position S242, preferably a S242Q substitution.

95. The method of any one of paragraphs 85-94, wherein the bacillus stearothermophilus alpha-amylase has a substitution at position E188, preferably an E188P substitution.

96. The method of any one of paragraphs 85-95, wherein the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ Having a T1/2 (min) of at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as between 10-70, such as between 15-70, such as between 20-70, such as between 25-70, such as between 30-70, such as between 40-70, such as between 50-70, such as between 60-70.

97. The method of any one of paragraphs 85-96, wherein the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants having the following mutations in addition to I181 x + G182 x and optionally N193F:

98. the method of any one of paragraphs 85-97, wherein the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

-I181 +g182 +n193F +v59A Q r+e129V +k177L +r179E +q254S +m284V; and

99. The method of any one of paragraphs 91-98, wherein the alpha-amylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID NO:1 herein.

100. The method of any one of paragraphs 85-98, wherein the alpha-amylase is a bacillus licheniformis alpha-amylase, or variant thereof.

101. The method of paragraph 100 wherein the Bacillus licheniformis alpha-amylase is one of the SEQ ID NOs as shown herein as 21.

102. The method of any one of paragraphs 100-101, wherein the alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% identity to the mature portion of the polypeptide of SEQ ID No. 21 herein.

103. The method of any of paragraphs 85-102, wherein the alpha-amylase may be present and/or added at a concentration of 0.1-100 micrograms/gram DS, e.g., 0.5-50 micrograms/gram DS, e.g., 1-25 micrograms/gram DS, e.g., 1-10 micrograms/gram DS, e.g., 2-5 micrograms/gram DS.

104. The method of any one of paragraphs 85-103, wherein during liquefaction, from 1-10 micrograms of thermolysin and 1-10 micrograms of bacillus stearothermophilus alpha-amylase are present and/or added.

105. The process of any one of paragraphs 85-104, wherein glucoamylase is present and/or added in liquefaction step i).

106. The method of paragraph 105, wherein the glucoamylase present and/or added during liquefaction has a thermostability of at least 20%, e.g., at least 30%, preferably at least 35% at 85 ℃, pH 5.3.

107. The method of paragraph 105 or 106, wherein the glucoamylase has a relative activity of at least 90%, preferably at least 95%, preferably at least 97% at a pH optimum of pH 5.0.

108. The method of any one of paragraphs 105-102, wherein the glucoamylase has a pH stability of at least 80%, at least 85%, at least 90% at pH 5.0.

109. The method of any one of paragraphs 105-103, wherein the glucoamylase present and/or added during liquefaction step i) is derived from a penicillium strain, in particular a penicillium oxalate strain as disclosed in WO 2011/127802 as SEQ ID No. 2 or as SEQ ID No. 9 or 14 herein.

110. The method of paragraphs 105-109, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the mature polypeptide shown in SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 9 or 14 herein.

111. The method of any one of paragraphs 105-110, wherein the glucoamylase is a variant of the penicillium oxalicum glucoamylase of SEQ ID No. 2 shown in WO 2011/127802 having a K79V substitution (numbering using the mature sequence shown in SEQ ID No. 14), e.g., the variant disclosed in WO 2013/053801.

112. The method of any one of paragraphs 105-111, wherein the penicillium oxalicum glucoamylase has a K79V substitution (numbered using SEQ ID NO: 14) and further has one of:

T65A; or (b)

Q327F; or (b)

E501V; or (b)

Y504T; or (b)

Y504; or (b)

t65a+q327F; or (b)

t65a+e501V; or (b)

t65a+y504T; or (b)

T65a+y504; or (b)

Q327F+E501V; or (b)

Q327F+Y504T; or (b)

Q327 f+y504; or (b)

E501V+Y504T; or (b)

E501 v+y504; or (b)

T65a+q327f+e501V; or (b)

T65a+q327f+y504T; or (b)

T65a+e501v+y504T; or (b)

Q327F+E501V+Y504T; or (b)

T65a+q327 f+y504; or (b)

T65a+e501 v+y504; or (b)

Q327f+e501 v+y504; or (b)

T65a+q327f+e501v+y504T; or (b)

T65A+Q327F+E501V+Y504*；

E501V+Y504T; or (b)

t65a+k161S; or (b)

t65a+q405T; or (b)

t65a+q327W; or (b)

t65a+q327F; or (b)

t65a+q327Y; or (b)

p11f+t65a+q327F; or (b)

R1k+d3w+k5q+g7v+n8s+t10k+p1s+t65a+q 327F; or (b)

p2n+p4s+p11f+t65a+q327F; or (b)

p11f+d26c+k33c+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

R1e+d3n+p4g+g6r+g7a+n8a+t10d+p11d+t65a+q327F; or (b)

p11f+t65a+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P11f+t65a+q327w+e501v+y504T; or (b)

T65a+q327f+e501v+y504T; or (b)

t65a+s105p+q327W; or (b)

t65a+s105p+q327F; or (b)

T65a+q327w+s364P; or (b)

T65a+q327f+s364P; or (b)

t65a+s103n+q327F; or (b)

P2n+p4s+p11f+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447S; or (b)

P2n+p4s+p11f+t65a+i172v+q327F; or (b)

P2n+p4s+p11f+t65a+q327 f+n502; or (b)

P2n+p4s+p11f+t65a+q327f+n502t+p563s+k571E; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+n564d+k571S; or (b)

P2n+p4s+p11f+t65a+q327f+s377t; or (b)

P2n+p4s+p11f+t65a+v325t+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+i172v+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s377t+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+i375 a+e501 v+y504T; or (b)

P2n+p4s+p11f+t65a+k21a+k217d+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

P2n+p4s+t10d+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+f12y+t65a+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t568N; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+k524t+g526A; or (b)

P2n+p4s+p11f+k34y+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+f80+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k12s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+n502 t+y504; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+k9a+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9g+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9i+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9l+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+l72v+q327f+e501v+y504T; or (b)

S255n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+e7n+v79 k+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+g220n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+y245n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q257n+q7f+e501 v+y504T; or (b)

P2n+p4s+p11f+t65a+d279n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s359n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+d370n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460t+p468t+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+t463n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s465n+e501v+y504T; or (b)

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T。

113. The method of any one of paragraphs 105-112, wherein the glucoamylase present and/or added in liquefaction is a penicillium oxalicum glucoamylase having a K79V substitution (numbered with SEQ ID NO: 14) and further having one of:

-P11F T65A Q327F

P2N P4S P11F T65A Q327F (numbering using SEQ ID NO: 14).

114. The method of any one of paragraphs 105-113, wherein the glucoamylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 14 herein.

115. The method of any one of paragraphs 85-114, further wherein glucoamylase is present and/or added during saccharification and/or fermentation.

116. The method of paragraph 115, wherein the glucoamylase present and/or added during saccharification and/or fermentation is of fungal origin, preferably from a strain of aspergillus, preferably a strain of aspergillus niger, aspergillus awamori, or aspergillus oryzae; or a strain of Trichoderma, preferably a strain of Trichoderma reesei; or a strain of the genus panus, preferably panus emersonii; or a strain of midwifery; or a strain of the genus Myxophyllum, for example, myxophyllum palustris or Myxophyllum, or a strain of the genus Nicotiana.

117. The method of any one of paragraphs 115-116, wherein the glucoamylase is derived from eimeria gracilis (e.g., one of SEQ ID nos. 19 shown herein).

118. The method of any one of paragraphs 115-117, wherein the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 19 herein;

119. The method of any of paragraphs 115-119, wherein the glucoamylase is derived from Philippine (e.g., one of SEQ ID NOs: 15 as set forth herein).

120. The method of any one of paragraphs 115-119, wherein the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 15 herein;

121. The method of any of paragraphs 115-120, wherein the glucoamylase is derived from Philippine brown fungus (e.g., one of SEQ ID NOs: 17 as set forth herein).

122. The method of any one of paragraphs 115-121, wherein the glucoamylase is selected from the group consisting of:

(i) A glucoamylase comprising the mature polypeptide of SEQ ID No. 17 herein;

123. The method of any of paragraphs 115-122, wherein glucoamylase is present in combination with alpha-amylase during saccharification and/or fermentation.

124. The method of paragraph 123 wherein the alpha-amylase present during saccharification and/or fermentation is of fungal or bacterial origin.

125. The method of paragraph 123 or 124, wherein the alpha-amylase present and/or added in saccharification and/or fermentation is derived from a rhizomucor strain, preferably a strain of rhizomucor minium, such as one of SEQ ID nos. 3 shown in WO 2013/006756, such as a rhizomucor minium alpha-amylase hybrid having an aspergillus niger linker and a starch binding domain, such as one of SEQ ID nos. 16.

126. The method of any one of paragraphs 123-125, wherein the alpha-amylase present in saccharification and/or fermentation is selected from the group consisting of:

(i) An alpha-amylase comprising the mature polypeptide of SEQ ID NO. 16 herein;

127. The method of any one of paragraphs 123-126, wherein the alpha-amylase is an alpha-amylase variant shown in SEQ ID No. 13 having at least one of the following substitutions or combinations: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; s123h+y141W; g20s+y141W; a76g+y141W; g128d+y141W; g128d+d143N; p219C+Y141W; n142d+d143N; y141w+k192R; y141w+d143N; y141w+n383R; y141w+p219c+a265C; y141 w+n517d+d143N; y141w+k192R V410A; g128d+y141w+d143N; y141w+d143n+p219C; y141w+d143n+k192R; g128d+d143n+k192R; y141w+d143 n+k168r+p219C; g217d+y141 w+d143n+k192R; or G128D+Y141W+D143N+K192R+P219C (numbered with SEQ ID NO: 16).

128. The method of any one of paragraphs 123-127, wherein the alpha-amylase is derived from rhizomucor parvum having an aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD), preferably as disclosed in SEQ ID No. 16 herein, preferably with one or more of the following substitutions: G128D, D N, preferably G128D+D143N (numbered using SEQ ID NO: 16).

129. The method of any one of paragraphs 123-128, wherein the alpha-amylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID NO:16 herein.

130. The method of any one of paragraphs 85-129, further wherein pullulanase is present and/or added during liquefaction and/or saccharification and/or fermentation.

131. The composition of paragraph 130, wherein the pullulanase is a GH57 family pullulanase, wherein the pullulanase preferably comprises the X47 domain as disclosed in WO 2011/087836.

132. The method of paragraphs 130-131, wherein the pullulanase is derived from a strain from the genus thermophilic, including pyrococcus thermophilus and pyrococcus hydrothermal, or hybrids thereof.

133. The method of any one of paragraphs 130-132, wherein the pullulanase is a hot water pyrococcus pullulanase truncated at position X4 or a hot water pyrococcus/thermophilic coccus hybrid enzyme having a truncation position X4, disclosed in WO 2011/087836 or shown in SEQ ID No. 12 herein.

134. The method of any one of paragraphs 85-133, further comprising, prior to liquefying step i), the steps of:

ii) forming a slurry comprising the starch-containing material and water.

135. The method of any one of paragraphs 85-134, wherein at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material is suitable for passing through a screen having a #6 screen.

136. The method of any one of paragraphs 85-135, wherein the pH during liquefaction is between above 4.5-6.5, such as about 4.8, or between 5.0-6.2, such as 5.0-6.0, such as between 5.0-5.5, such as about 5.2, such as about 5.4, such as about 5.6, such as about 5.8.

137. The method of any one of paragraphs 85-136, wherein the temperature during liquefaction is above the initial gelatinization temperature, e.g., in the range from 70-100 ℃, e.g., between 75-95 ℃, e.g., between 75-90 ℃, preferably between 80-90 ℃, especially about 85 ℃.

138. The method of any one of paragraphs 85-137, wherein a jet cooking step is performed prior to liquefaction in step a).

139. The method of paragraph 138, wherein the jet cooking is carried out at a temperature of 110 ℃ to 145 ℃, preferably 120 ℃ to 140 ℃, e.g. 125 ℃ to 135 ℃, preferably about 130 ℃ for about 1 to 15 minutes, preferably for about 3 to 10 minutes, especially about 5 minutes or so.

140. The method of any one of paragraphs 85-139, wherein saccharification and fermentation are conducted sequentially or simultaneously.

141. The method of any of paragraphs 85-140, wherein saccharification is conducted at a temperature of from 20 ℃ -75 ℃, preferably from 40 ℃ -70 ℃, e.g., about 60 ℃, and at a pH of between 4 and 5.

142. The method of any one of paragraphs 85-141, wherein fermentation or Simultaneous Saccharification and Fermentation (SSF) is conducted at a temperature of from 25 ℃ to 40 ℃, e.g., from 28 ℃ to 35 ℃, e.g., from 30 ℃ to 34 ℃, preferably about 32 ℃. In one embodiment, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.

143. The method of any one of paragraphs 85-142, wherein the fermentation product is recovered after fermentation, e.g., by distillation.

144. The method of any one of paragraphs 85-143, wherein the fermentation product is an alcohol, preferably ethanol, especially fuel ethanol, potable ethanol, and/or industrial ethanol.

145. The method of any one of paragraphs 85-144, wherein the starch-containing starting material is whole grain.

146. The method of any one of paragraphs 85-145, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, tapioca, manioc, tapioca, sorghum, rice or potato.

147. The method of any one of paragraphs 85-146, wherein the fermenting microorganism is a yeast, preferably a saccharomyces strain, especially a saccharomyces cerevisiae strain.

148. The method of any one of paragraphs 85-147, wherein the alpha-amylase is a bacterial or fungal alpha-amylase.

149. The method of any of paragraphs 85-148, comprising the steps of:

-an alpha-amylase derived from bacillus stearothermophilus;

-optionally a penicillium oxalicum glucoamylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

150. The method of paragraphs 85-149, comprising the steps of:

an alpha-amylase, preferably derived from Bacillus stearothermophilus, at pH 4.5, 85℃and 0.12mM CaCl ₂ T1 x 2 (min) with at least 10 below;

-optionally a glucoamylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

151. The method of paragraphs 85-150, comprising the steps of:

-a penicillium oxalicum glucoamylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

152. The method of paragraphs 85-151, comprising the steps of:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbered with SEQ ID NO: 14);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

153. The method of paragraphs 85-152, comprising the steps of:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

greater than 2 micrograms, for example between 2 and 5 micrograms, preferably about 3 micrograms of intense fireball protease per gram of DS Dry Solids (DS);

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbered with SEQ ID NO: 14);

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

154. The method of paragraphs 85-153, comprising the steps of:

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

greater than 2 micrograms, for example between 2 and 5 micrograms, preferably about 3 micrograms of intense fireball protease per gram of DS Dry Solids (DS)

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbered with SEQ ID NO: 14);

c) Fermentation is performed using a fermenting organism.

155. The method of any one of paragraphs 85-154, wherein the ratio between alpha-amylase and glucoamylase during liquefaction is between 1:1 and 1:10, e.g., about 1:2 (micrograms alpha-amylase/g DS: micrograms glucoamylase/g DS).

156. The method of any one of paragraphs 85-155, wherein the ratio between alpha-amylase and protease during liquefaction is between 1:1 and 1:25, e.g., in the range between 1:1.2 and 1:10, e.g., about 1:1.4 (micrograms of alpha-amylase/gram of DS: micrograms of protease/gram of DS).

157. An enzyme composition comprising:

i) Bacillus species alpha-amylase, or a variant thereof;

ii) a strong fireball protease;

158. The enzyme composition of paragraph 157, wherein the ratio between alpha-amylase and protease is between 1:1.2 and 1:10, e.g., in the range of about 1:1.4 (micrograms of alpha-amylase: micrograms of protease).

159. The enzyme composition of paragraphs 157-158, wherein the enzyme composition comprises glucoamylase and the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase: micrograms of glucoamylase).

160. The enzyme composition of any one of paragraphs 157-159, wherein the alpha-amylase is a bacterial or fungal alpha-amylase.

161. The enzyme composition of any one of paragraphs 157-160, wherein the alpha-amylase is from a bacillus, e.g., a bacillus stearothermophilus strain, particularly a variant of a bacillus stearothermophilus alpha-amylase, e.g., one of SEQ ID No. 3 or 1 set forth herein in WO 99/019467.

162. The enzyme composition of any one of paragraphs 157-161, wherein the bacillus stearothermophilus alpha-amylase or variant thereof is truncated, preferably truncated to have about 491 amino acids, e.g., from 480-495 amino acids.

163. The enzyme composition of any one of paragraphs 157-162, wherein the bacillus stearothermophilus alpha-amylase has a double deletion, preferably at position I181+ G182, and optionally has an N193F substitution, or a double deletion of R179 and G180 (numbered using SEQ ID NO: 1).

164. The enzyme composition of any one of paragraphs 157-163, wherein the bacillus stearothermophilus alpha-amylase has a substitution at position S242, preferably a S242Q substitution.

165. The enzyme composition of any one of paragraphs 157-164, wherein the bacillus stearothermophilus alpha-amylase has a substitution at position E188, preferably an E188P substitution.

166. The enzyme composition of any one of paragraphs 157-165, wherein the alpha-amylase is at pH 4.5, 85 ℃, 0.12mM CaCl ₂ Having a T1/2 (min) of at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as between 10-70, such asBetween 15 and 70, for example between 20 and 70, for example between 25 and 70, for example between 30 and 70, for example between 40 and 70, for example between 50 and 70, for example between 60 and 70.

167. The enzyme composition of any one of paragraphs 157-166, wherein the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants having the following mutations:

-I181*+G182*+N193F+E129V+K177L+R179E；

-I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-I181 +g182 +n193F +v59A Q r+e129V +k177L +r179E +q254S +m284V; and

168. The enzyme composition of any one of paragraphs 157-167, wherein the alpha-amylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID NO:1 herein.

169. The enzyme composition of any one of paragraphs 157-168, wherein the alpha-amylase is a bacillus licheniformis alpha-amylase, or variant thereof.

170. The enzyme composition of paragraph 169, wherein the bacillus licheniformis alpha-amylase is one of the SEQ ID nos. 21 shown herein.

171. The enzyme composition of any one of paragraphs 157-170, wherein the alpha-amylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% identity to the mature portion of the polypeptide of SEQ ID No. 21 herein.

172. The enzyme composition of any one of paragraphs 157-171, wherein the enzyme composition comprises bacillus licheniformis alpha-amylase and a thermolysin.

173. The enzyme composition of any one of paragraphs 157-172, wherein the enzyme composition further comprises a glucoamylase.

174. The composition of any one of paragraphs 157 or-173, wherein the pyrococcus furiosus is one of the SEQ ID NOs 13 shown herein.

175. The composition of any one of paragraphs 157-174, wherein the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

176. The composition of any one of paragraphs 157-175, wherein the enzyme composition further comprises a glucoamylase shown in SEQ ID No. 14, or a variant thereof.

177. The composition of paragraphs 152-166, wherein the glucoamylase has a thermostability of at least 20%, e.g., at least 30%, preferably at least 35%, at 85 ℃ and pH 5.3.

178. The composition of any one of paragraphs 176-177, wherein the glucoamylase has a relative activity of at least 90%, preferably at least 95%, preferably at least 97% at a pH optimum of pH 5.0.

179. The composition of any one of paragraphs 176-178, wherein the glucoamylase has a pH stability of at least 80%, at least 85%, at least 90% at pH 5.0.

180. The composition of any one of paragraphs 176-179, wherein the glucoamylase is derived from a penicillium strain, particularly a penicillium oxalicum strain, disclosed as SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 9 or 14 herein.

181. The composition of paragraphs 176-170, wherein the glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the mature polypeptide shown in SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 9 or 14 herein.

182. The composition of any one of paragraphs 176-181, wherein the glucoamylase is a variant of the penicillium oxalicum glucoamylase disclosed as SEQ ID No. 2 in WO 2011/127802 or SEQ ID No. 14 herein having a K79V substitution (numbered using the mature sequence shown in SEQ ID No. 14), e.g., the variant disclosed in WO 2013/053801.

183. The composition of any one of paragraphs 176-182, wherein the penicillium oxalicum glucoamylase has a K79V substitution (numbered using SEQ ID NO: 14) and further has one of:

T65A; or (b)

Q327F; or (b)

E501V; or (b)

Y504T; or (b)

Y504; or (b)

t65a+q327F; or (b)

t65a+e501V; or (b)

t65a+y504T; or (b)

T65a+y504; or (b)

Q327F+E501V; or (b)

Q327F+Y504T; or (b)

Q327 f+y504; or (b)

E501V+Y504T; or (b)

E501 v+y504; or (b)

T65a+q327f+e501V; or (b)

T65a+q327f+y504T; or (b)

T65a+e501v+y504T; or (b)

Q327F+E501V+Y504T; or (b)

T65a+q327 f+y504; or (b)

T65a+e501 v+y504; or (b)

Q327f+e501 v+y504; or (b)

T65a+q327f+e501v+y504T; or (b)

T65A+Q327F+E501V+Y504*；

E501V+Y504T; or (b)

t65a+k161S; or (b)

t65a+q405T; or (b)

t65a+q327W; or (b)

t65a+q327F; or (b)

t65a+q327Y; or (b)

p11f+t65a+q327F; or (b)

R1k+d3w+k5q+g7v+n8s+t10k+p1s+t65a+q 327F; or (b)

p2n+p4s+p11f+t65a+q327F; or (b)

p11f+d26c+k33c+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

R1e+d3n+p4g+g6r+g7a+n8a+t10d+p11d+t65a+q327F; or (b)

p11f+t65a+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P11f+t65a+q327w+e501v+y504T; or (b)

T65a+q327f+e501v+y504T; or (b)

t65a+s105p+q327W; or (b)

t65a+s105p+q327F; or (b)

T65a+q327w+s364P; or (b)

T65a+q327f+s364P; or (b)

t65a+s103n+q327F; or (b)

P2n+p4s+p11f+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447S; or (b)

P2n+p4s+p11f+t65a+i172v+q327F; or (b)

P2n+p4s+p11f+t65a+q327 f+n502; or (b)

P2n+p4s+p11f+t65a+q327f+n502t+p563s+k571E; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+n564d+k571S; or (b)

P2n+p4s+p11f+t65a+q327f+s377t; or (b)

P2n+p4s+p11f+t65a+v325t+q327W; or (b)

P2n+p4s+p11f+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+i172v+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s377t+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327F; or (b)

P2n+p4s+p11f+t65a+q327f+i375a+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k21a+k217d+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

P2n+p4s+t10d+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+f12y+t65a+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+t10e+e18n+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t568N; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+k524t+g526A; or (b)

P2n+p4s+p11f+k34y+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+r31s+k33v+t65a+q327f+d445n+v447s+e501v+y504T; or (b)

P2n+p4s+p11f+d26n+k34y+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+f80+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k12s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+n502 t+y504; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+s103n+q327f+e501v+y504T; or (b)

K5a+p11f+t65a+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+e501v+y504t+t516p+k524t+g526A; or (b)

P2n+p4s+p11f+t65a+k9a+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9g+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9i+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9l+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+k9s+q327 f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+l72v+q327f+e501v+y504T; or (b)

S255n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+e7n+v79 k+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+g220n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+y245n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q257n+q7f+e501 v+y504T; or (b)

P2n+p4s+p11f+t65a+d279n+q327f+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s359n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+d370n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460s+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+v460t+p468t+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+t463n+e501v+y504T; or (b)

P2n+p4s+p11f+t65a+q327f+s465n+e501v+y504T; or (b)

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T。

184. The composition of any one of paragraphs 176-183, wherein the glucoamylase is a penicillium oxalicum glucoamylase having a K79V substitution (numbered using SEQ ID NO: 14) and further having one of the following substitutions:

-P11F+T65A+Q327F

P2N+P4S+P11F+T65A+Q327F (numbering using SEQ ID NO: 14).

185. The composition of any of paragraphs 182-184, wherein the glucoamylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, e.g., even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 14 herein.

186. The composition of any one of paragraphs 157-185, further comprising a pullulanase.

187. The composition of paragraph 186, wherein the pullulanase is a GH57 family pullulanase, wherein the pullulanase preferably comprises the X47 domain as disclosed in WO 2011/087836.

188. The method of paragraphs 186-187, wherein the pullulanase is derived from a strain from the genus thermophilic, including pyrococcus thermophilus and pyrococcus hydrothermal, or hybrids thereof.

189. The composition of any one of paragraphs 186-188, wherein the pullulanase is a hot water pyrococcus pullulanase truncated at position X4 or a hot water pyrococcus/thermophilic coccus hybrid enzyme disclosed in WO 2011/087836 or shown in SEQ ID No. 12 herein having a truncation position X4.

190. The composition of any one of paragraphs 157-189, comprising

-bacillus stearothermophilus alpha-amylase, or a variant thereof;

-a strong fireball protease; and

the enzyme Penicillium oxalicum glucoamylase,

191. The composition of any one of paragraphs 157-190, comprising

Alpha-amylase, preferably derived from Bacillus stearothermophilus, at pH 4.5, 85℃and 0.12mM CaCl ₂ T1/2 (min) with at least 10;

-a strong fireball protease; and

the enzyme Penicillium oxalicum glucoamylase,

192. The composition of any one of paragraphs 157-191, comprising

-E129V+K177L+R179E；

-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；

-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V；

e129V+K177L+R179E+K220P+N224 L+S24Q+Q254S (numbered with SEQ ID NO:1 herein);

-a strong fireball protease; and

-K79V；

-k79v+p11f+t65a+q327F; or (b)

-k79 v+p2n+p4s+p1f+t65a+q 327F; or (b)

-k7v+p11f+d26c+k33c+t65a+q 327F; or (b)

-k79v+p2n+p4s+p11f+t65a+q327w+e501v+y504T; or (b)

-k79v+p2n+p4s+p11f+t65a+q327f+e501v+y504T; or (b)

K79 V+P1F+T65A+Q327 W+E501V+Y504T (numbering using SEQ ID NO: 14).

193. The enzyme composition of any one of paragraphs 190-192, wherein the ratio between alpha-amylase and protease is in the range of between 1:1.2 and 1:10, e.g., about 1:1.4 (micrograms of alpha-amylase: micrograms of protease).

194. The enzyme composition of any one of paragraphs 190-193, wherein the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase: micrograms of glucoamylase).

Claims

-an alpha-amylase;

b) Saccharification using a glucoamylase;

c) Fermenting using a fermenting organism;

d) Recovering the fermentation product to form distillers grains;

e) Separating the distillers grains into thin stillage and wet cake;

f) Optionally concentrating the thin stillage into a slurry;

wherein the oil is recovered from:

-starch-containing material liquefied after step a); and/or

Downstream of the fermentation step c).

2. The method of claim 1, wherein during and/or after liquefying the starch-containing material; from the whole stillage; from the thin stillage; or recovering oil from the slurry.

3. The method according to any one of claims 1-2, wherein 0.5-100 microgram of pyrococcus furiosus protease per gram DS, such as 1-50 microgram of pyrococcus furiosus protease per gram DS, such as 1-10 microgram of pyrococcus furiosus protease per gram DS, such as 1.5-5 microgram of pyrococcus furiosus protease per gram DS, such as about or more than 1.5 microgram of pyrococcus furiosus protease per gram DS is present and/or added in liquefaction step a).

4. The method of any one of claims 1-8, wherein the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

5. The process of any one of claims 1-4, wherein in steps a) -c), no nitrogen compounds are present and/or added, e.g. during saccharification step b), fermentation step c), or Simultaneous Saccharification and Fermentation (SSF).

6. A method for producing a fermentation product from starch-containing material, the method comprising the steps of:

-an alpha-amylase;

b) Saccharification using a glucoamylase;

c) Fermentation is performed using a fermenting organism.

7. The method of claim 6, wherein 2-100 micrograms/gram DS, such as 2.5-50 micrograms/gram DS, such as 2.5-10 micrograms/gram DS, such as 2.5-5 micrograms/gram DS, especially about 3 micrograms/gram DS, is a thermolysin.

8. The method of any one of claims 6-7, wherein the strong fireball protease is a protease having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 13 herein.

9. The method of any one of claims 6-8, wherein in steps a) -c), no nitrogen compounds are present and/or added, e.g. during saccharification step b) or fermentation step c) or Simultaneous Saccharification and Fermentation (SSF).

10. An enzyme composition, the enzyme composition comprising:

i) Bacillus species alpha-amylase, or a variant thereof;

ii) a strong fireball protease;

11. The enzyme composition according to claim 10, wherein the ratio between alpha-amylase and protease is in the range between 1:1.2 and 1:10, such as about 1:1.4 (micrograms of alpha-amylase: micrograms of protease).

12. The enzyme composition of claims 10-11, wherein the enzyme composition comprises glucoamylase and the ratio between alpha-amylase and glucoamylase is between 1:1 and 1:10, e.g., about 1:2 (micrograms of alpha-amylase: micrograms of glucoamylase).

13. The composition of any one of claims 157-189, comprising

-bacillus stearothermophilus alpha-amylase, or a variant thereof;

-a strong fireball protease; and

the enzyme Penicillium oxalicum glucoamylase,

14. The composition of any one of claims 10-13, comprising

Alpha-amylase, preferably derived from Bacillus stearothermophilus, at pH 4.5, 85℃and 0.12mM CaCl ₂ T having at least 10 ¹ / ₂ (min)；

-a strong fireball protease; and

the enzyme Penicillium oxalicum glucoamylase,